WO2004056986A2

WO2004056986A2 - Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors

Info

Publication number: WO2004056986A2
Application number: PCT/NL2003/000909
Authority: WO
Inventors: Arie Pieter Otte; Theodorus Hendrikus Jacobus Kwaks; Richard George Antonius Bernardus Sewalt
Original assignee: Chromagenics BV
Current assignee: Chromagenics BV
Priority date: 2002-12-20
Filing date: 2003-12-19
Publication date: 2004-07-08
Anticipated expiration: 2005-06-20
Also published as: US8263393B2; EP1572994B1; AU2003290453A8; ATE354645T1; EP1572994A2; DK1572994T3; AU2003290453A1; DE60312039T2; US20060003416A1; WO2004056986A3; DE60312039D1

Abstract

The invention is concerned with means and methods for providing a cell with a protein expression unit comprising providing a nucleic acid comprising said unit with a nucleic acid encoding a binding site for a member of a chromatin modification system for rendering chromatin more accessible for transcription (opener), wherein said opener is present in said cell. Preferred openers comprise histone modification proteins, chromatin remodeling proteins and trithorax group proteins or equivalents. The cells thus generated and nucleic acids encoding such openers are provided. Openers are preferred in the context of STAR and TRAP sequences.

Description

Title: Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors.

The invention relates to the fields of biochemistry, molecular biology and pharmacology. More specifically the present invention relates to the production of proteins in a host cell. In one embodiment the invention relates methods for improving the predictability, yield and/or stability of the production of proteins in a (host) cell. Methods herein are suited for production of one or more proteins.

Proteins are produced in systems for a wide range of applications in biology and biotechnology. These include research into cellular and molecular function, production of proteins as biopharmace ticals or diagnostic reagents, and modification of the traits or phenotypes of livestock and crops.

Biopharmaceuticals are usually proteins that have an extracellular function, such as antibodies for immunotherapy or hormones or cytokines for eliciting a cellular response. Proteins with extracellular functions exit the cell via a secretory pathway, and undergo post-translational modifications during secretion (Chevet et al 2001) The modifications (primarily glycosylation and disulfi.de bond formation) do not naturally occur in bacteria. Moreover, the specific oligosaccharides attached to proteins by glycosylating enzymes are typically species and cell-type specific. These considerations often limit the choice of host cells for heterologous protein production to eukaryotic cells (Kaufman, 2000). For expression of human therapeutic proteins, host cells such as bacteria, yeast, or plants may be inappropriate. Even the subtle differences in protein glycosylation between rodents and human, for example, can be sufficient to render proteins produced in rodent cells unacceptable for therapeutic use (Sheeley et al., 1997). The consequences of improper (i.e. non- human) glycosylation include immuno genicity, reduced functional half-life, and loss of activity. For proteins where this is a problem the choice of host cells is limited further, to human cell lines or to cell lines such as Chinese Hamster Ovary (CHO) cells, which may produce glycoproteins with human-like carbohydrate structures (Liu, 1992).

Some proteins of biotechnological interest are functional as multimers, i.e. they consist of two or more, possibly different, polypeptide chains in their biologically and/or biotechnologically active form. Examples include antibodies (Wright & Morrison, 1997), bone morphogenetic proteins (Groeneveld & Burger, 2000), nuclear hormone receptors (Aranda & Pascual, 2001), heterodimeric cell surface receptors (e.g., T cell receptors, (Chan & Mak, 1989)), integrins (Hynes, 1999), and the glycoprotein hormone family (chorionic gonadotrophin, pituitary luteinizing hormone, follicle -stimulating hormone, and thyroid-stimulating hormone, (Thotakura & Blithe, 1995)). Production of such multimeric proteins in heterologous systems is technically difficult due to a number of limitations of current expression systems. These limitations include (1) difficulties in isolating recombinant cells/cell lines that produce the monomer polypeptides at high levels (predictability and yield), and (2) declines in the levels of expression during the industrial production cycle of the proteins (stability). These problems are described in more detail below.

(1) Recombinant proteins such as antibodies that are used as therapeutic compounds need to be produced in large quantities. The host cells used for recombinant protein production must be compatible with the scale of the industrial processes that are employed. Specifically, the transgene (or the gene encoding a protein of interest, the two terms are used interchangeably herein) expression system used for the heterologous protein needs to be retained by the host cells in a stable and active form during the growth phases of scale-up and production. This is achieved by integration of the transgene into the genome of the host cell. However, creation of recombinant cell lines by conventional means is a costly and inefficient process due to the unpredictability of transgene expression among the recombinant host cells. The unpredictability stems from the high likelihood that the transgene will become inactive due to gene silencing (McBurney et al., 2002). Using conventional technologies, the proportion of recombinant host cells that produce one polypeptide at high levels ranges from 1-2%. In order to construct a cell line that produces two polypeptides at high levels, the two transgenes are generally integrated independently. If the two transgenes are transfected simultaneously on two separate plasmids, the proportion of cells that will produce both polypeptides at high levels will be the arithmetic product of the proportions for single transgenes. Therefore the proportion of such recombinant cell Hnes ranges from one in 2,500 to one in 10,000. For multimeric proteins with three or more subunits, the proportions decline further. These high-producing cell hnes must subsequently be identified and isolated from the rest of the population. The methods required to screen for these rare high-expressing cell hnes are time-consuming and expensive.

An alternative to simultaneous transfection of two transgene-bearing plasmids is sequential transfection. In this case the proportion of high-yielding clones will be the sum of the proportions for single transgenes, i.e. 2-4%.

Sequential transfection however has (major) drawbacks, including high costs and poor stability. The high costs results from various factors: in particular, the time and resources required for screening for high-expressing cell hnes is doubled, since high expression of each subunit must be screened for separately. The poor overall stability of host cells expressing two polypeptides is a consequence of the inherent instability of each of the two transgenes. (2) Silencing of transgene expression during prolonged host cell cultivation is a commonly observed phenomenon. In vertebrate cells it can be caused by formation of heterochromatin at the transgene locus, which prevents transcription of the transgene. Transgene silencing is stochastic; it can occur shortly after integration of the transgene into the genome, or only after a number of cell divisions. This results in heterogeneous cell populations after prolonged cultivation, in which some cells continue to express high levels of recombinant protein while others express low or undetectable levels of the protein (Martin & Whitelaw, 1996, McBurney et al., 2002). A cell line that is used for heterologous protein production is derived from a single cell, yet is often scaled up to, and maintained for long periods at, cell densities in excess of ten million cells per millilitre in cultivators of 1,000 litres or more. These large cell populations (10¹⁴ - 10¹⁶ cells) are prone to serious declines in productivity due to transgene silencing (Mighaccio et al., 2000, Strutzenberger et al., 1999).

The instability of expression of recombinant host cells is particularly severe when transgene copy numbers are amplified in an attempt to increase yields. Transgene amplification is achieved by including a selectable marker gene such as dihydrofolate reductase (DHFR) with the transgene during integration (Kaufman 2000). Increased concentrations of the selection agent (in the case of DHFR, the drug methotrexate) select for cells that have amplified the number of DHFR genes in the chromosome (Kaufman and Sharp 1982). Since the transgene and DHFR are eo-localized in the chromosome, the transgene copy number increases too. This is correlated with an increase in the yield of the heterologous protein (Kaufman, 1990). However, the tandem repeats of transgenes that result from amplification are highly susceptible to silencing (Garrick et al., 1998, Kaufman, 1990, McBurney et al., 2002).

The above stated problems associated with conventional transgene expression technologies for protein production clearly demonstrate a need in the art for systems that overcomes these problems. Specifically, there is a need for expression systems that i) provide high predictability of expression, allowing balanced expression of multiple chains, ii) provide high yields, and iii) provide stability during an extended period during which the protein needs to be produced in large quantities, iv) result in an increased number of clones with appropriate expression levels. The present invention provides means and methods for improving characteristics of protein production in a cell. It has among others been found that chromatin modification systems for rendering chromatin more accessible for transcription have a pronounced effect on expression characteristics of protein expression when allowed to act thereon. In one embodiment the invention therefore provides a method for providing a cell with a protein expression unit comprising providing a nucleic acid comprising said unit with a nucleic acid encoding a binding site for a member of a chromatin modification system for rendering chromatin more accessible for transcription (opener), wherein said opener is present in said cell, said method further comprising providing said expression unit to said cell and culturing said cell to allow expression of said protein expression unit.

Histone modification systems have been shown to encompass proteins capable of rendering chromatin more accessible for transcription. An opener of the invention is therefore preferably a histone modification enzyme, preferably capable of modifying a N-terminal histone tail. Histone modification plays an important role in both chromatin-associated repression and chromatin- associated activation of gene expression. For instance, acetylation of specific lysines in histone H3 and H4 tails is an important parameter. Normally histones are very basic proteins that bind tightly to the acid DNA strands.

Addition of an acetyl group to the histone tails converts the basic histones into more neutrally charged proteins. This results in a less tight interaction between the basic histones and the acid DNA strands. Acetylation is therefore associated with making the chromatin more open or accessible for transcription factors. Histone acetyltransferases (HATs) that add acetyl groups to the histone tails are therefore preferred openers of the present invention. Preferred, embodiments of HAT openers are p300/CBP, P/CAF (Yang et al 1996), and/or CBP (Bannister and Kouzarides 1996) or a functional part, derivative and/or analogue thereof. However, even today more HAT proteins, comprising similar function are identified. Such HAT proteins are of course also part of the invention. HAT proteins are likely to act in the context of a multiprotein complex at least in part to allow for specificity of action to certain regions of the chromatin. The Trithorax group (TrxG) protein trithorax (trx) is part of a complex that is involved in keeping genes in the activated state. It is, therefore, not surprising that the multiprotein complex of which the trx protein is part, also contains a HAT protein (Petruk et al 2001).

Opposing the action of HAT proteins are histone deacetyltransferases (HDACs) (Taunton et al 1996) that remove acetyl groups from histone H3 and H4 tails. This makes the histones more basic, which results in tightening the interactions between the histones and the acid DNA strands. Hence HDACs are negative regulators of gene expression. Polycomb group (PcG) protein are associated with HDACs and the repression executed by PcG proteins is in part mediated through histone deacetylation (Van der Vlag and Otte 1999). The opposing activating and repressing roles of respectively TrxG and PcG proteins is thus reflected in the association of HAT and HDAC proteins to respectively TrxG and PcG proteins.

Also specific methylated histone tails have activity in opening chromatin according to the invention. Some types of methylation are associated with rendering chromatin more accessible for transcription, whereas other types of methylation are associated with rendering chromatin less accessible.

Methylation of lysine K9 and K27 of histone H3 hallmarks repressed states of chromatin. When H3 K9 is methylated, a docking place for the chromatin- associated repressor protein HP1 protein (heterochromatin protein 1) is created (Bannister et al 2001; Lachner et al 2001). When H3 K27 is methylated, a docking place for the chromatin/associated repressor protein Polycomb (Pc) is created (Muller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, Wild B, Miller EL, O'Connor MB, Kingston RE, Simon JA: Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 2002, 111 : 197-208.)

In contrast, the Ashl protein (Nakamura et al 2000) is a trithorax group proteins that acts as a positive regulator of gene expression. Ashl has methyltransferase activity and adds a methyl group to at least lysine K4 of histone H3 (Beisel et al 2002). A methyltransferase capable of adding a methyl group to at least lysine K4 of histone H3 is thus a preferred opener of the invention. Preferably, said opener comprises Ashl protein or a functional part, derivative and/or analogue thereof.

The opposing actions of the activating Trithorax group and repressing Polycomb group proteins can at least in part be explained by respectively histone acetylation versus histone deacetylation, but also in part by their differential abilities to methylate specific and distinct lysines in histone tails. Methylation of specific lysines in histone tails is often either be preceded or prevented by deacetylation of the same lysines, thereby creating a complex and hierarchical interplay between histone modifications. Beside the acetylation and methylation of histone tails also phosphate groups and ubiquitin groups can be added. Also these events can influence the order in which either acetylation or methylation of histone tails can take place. Collectively this complex interplay between histone modifications is referred to as the "histone code" that is considered as the most fundamental mechanistic explanation for both repressing and activating epigenetic gene regulation mechanisms. It is thus according to the invention entirely possible that among the histone phopsphorylating or ubiquinating enzymes there are those that can render chromatin more accessible to transcription.

The different histone methyltransferases have a structural protein motif, the SET domain in common. The SET domain (for Su(var)39, E(z) and trx, the three proteins in which the domain was first identified) is essential for histone methyltransferase activity to take place. It follows that targeting an activating histone methyltransferase or its functional part, the SET domain, can have a beneficial effect on gene expression by interfering at the level of chromatin structure.

Another preferred group of openers are comprised in the chromatin - remodelling proteins such as Tritorax group proteins, CHRAC proteins, ACF group proteins, and/or a NURF group protein. Polycomb group (PcG) mediated silencing is counteracted by gene-activating Trithorax group (TrxG) proteins. This has been estabhshed genetically in the fruit fly Drosophila where mutations in PcG and TrxG counteract each other (Kennison and Tamkun 1992). Also in other model systems, including man, PcG and TrxG complexes have been identified (Kingston et al 1996). Specific TrxG proteins are trithorax (Mazo et al, 1990; Petruk et al, 2001), trithorax-like (Farkas et al. 1994), Brahma (Tamkum et al 1992), ISWI (Elfring et al, 1994), Ashl (Nakamura et al, 2000; Beisel et al, 2002), moira (Crosby et al, 1999), and osa (Treisman et al, 1997).

One TrxG protein is Brahma (Tamkun et al 1992; Chiba et al 1994; Kal et al 2000; Sif et al 2001; Mizutani, 2002). This protein is part of a multimeric protein complex that operates as a so-called chromatin-remodelling complex. Chromatin-remodelling has been defined as the ATPase-dependent disruption of nucleosomes to facilitate binding of transcription factors to the chromatin (Kwon et al 1994; Imbalzano et al 1994; Quinn et al 1996). The chromatin becomes more open or accessible for transcription factors and thus transcription. Other chromatin-remodelling complexes have been defined, such as CHRAC (Varga-Weisz et al; 1997) and NURF (Tsukiyama and Wu 1995). A more comprehensive overview is given by Fyodorov and Kadonaga, 2001. Also these complexes operate in an ATPase dependent fashion. Thus in this embodiment the opener preferably comprises a chromatin-remodelling protein and preferably the Trithorax group protein Brahma, a CHRAC group protein, a NURF group protein, ACF group proteins, (for ATP-utilizing chromatin assembly and remodelling factor) (Ito et al 1997) or a functional part, derivative and/or analogue thereof. Purified ACF fractions contain Imitation SWI (ISWI) protein (Elfring et al, 1994). Three other proteins co-purify with this complex termed p47, pl70 and pl85 referring to their apparent molecular weight. In a preferred embodiment said chromatin-remodelling protein comprises an ISWI protein or a Brahma protein or a functional part, derivative and/or analogue thereof.

Trithorax group proteins have miscellaneous effects on chromatin, however, at least some proteins of the group are capable of rendering chromatin more accessible to transcription factors. Thus in a preferred embodiment said opener comprises a protein of the trithorax group and preferably comprises an ISWI protein or a trithorax protein, a trithorax-like protein, a Brahma protein, an Ash protein, a moira protein, an osa protein or a functional part, derivative and or analogue thereof

A functional part, derivative and/or analogue of an opener of the invention comprises the same activity in kind not necessarily in amount as an opener mentioned. This activity being a sequence specific nucleic acid binding activity specific for said binding site and a chromatin modification activity rendering chromatin more accessible for transcription. This chromatin modification activity may be intrinsic to said opener or may be present through enabling a further protein to act on said chromatin. Suitable parts may be generated by mutation, deletion and/or insertions of the opener. These may be tested in a method of the invention for functionality as an opener. Often parts of a protein can be identified that can be manipulated to at least some extend without affecting the kind of function of the protein. Such openers comprising such modifications are of course within the present invention. For openers that comprise the so-called SET domain, the functional part typically comprises this SET domain. Derivatives may be generated by for instance conservative amino acid substitutions. These typically retain the same function in kind. Analogues of openers of the present invention are typically proteins having the same or similar chromatin modification activity in kind not necessarily in amount. Suitable analogues may be found in other than the mentioned species. Such analogues can for instance be selected by amino acid and or nucleic acid homology. For instance ISWI2 has in humans the homologues BRGl and hbrm. ISWI2 is homologues to Brahma, whereas BAF170 and BAF155 are SWI3 homologues. Another non-limiting example of suitable homologues are BAF170, BAF155 and SWI3 which are homologues of moira. Such homologues are of course also part of the invention.

Many different proteins can acts as openers in the present invention. The opener may act directly on the accessibility of chromatin or indirectly via the association with a complex present in the cell, wherein the complex is instrumental in the accessibihty of the chromatin. An essential component of the opener of the present invention is the sequence specific association thereof with the binding site on the nucleic acid comprising the protein expression unit. The binding site may be a normal binding site for an opener. Alternatively, a binding specificity for said binding site is provided to an otherwise operable opener. In yet another embodiment a sequence specific nucleic acid binding specificity for said binding site is provided to a protein thereby resulting in an opener of the present invention. It is possible that proteins, when provided with a binding specificity for said binding site do not have a sequence specific binding specificity by themselves (i.e. prior to being provided with such specificity). Such proteins (further termed pre-openers) can be provided to the cell to achieve a generahzed effect on chromatin remodelling. This is another aspect of the present invention. The invention thus provides a cell comprising a protein expression unit, wherein said cell is provided with a pre-opener of the present invention. Such cells can, through the generahzed effect on chromatin re-modelling display favourable expression characteristics. This can for instance be due to a shift in the balance between activating and repressing complexes.

An opener may be expressed by the cell prior to providing the cell with the protein expression construct, for instance in case the cell naturally expresses said opener. Alternatively, the opener may be provided to the cell, for instance as a nucleic acid encoding the opener. When openers are used that have been provided with a specific nucleic acid binding activity toward the binding site, it is often appropriate to provide the cell with the opener. However, cell hnes may be created already expressing such opener. Such cell hnes can then subsequently be used to introduce protein expression unit of the invention at will. Cell hnes provided with a nucleic acid comprising an opener provided with a new sequence specific binding activity are therefore also part of the invention. Such cell hnes must of course carry the nucleic acid in stable form, thus preferably integrated into the genome of the cell. In a preferred embodiment such cell hnes are used for the collection of proteins produced through a means or a method of the invention. Preferred openers for such cell lines comprises HAT proteins provided with a new sequence specific binding activity. Preferably said HAT proteins comprise p300/CBP protein, a P/CAF protein, and/or a CBP protein or a functional part, derivative and/or analogue thereof. The new sequence specific binding activity preferably comprises a nucleic acid binding domain of a sequence specific DNA binding protein. Non- limiting examples are the GAL4 or the LexA DNA binding domains. However, many other sequence specific binding proteins can be used. A person skilled in the art can use DNA binding domains of a large number of different proteins and generate an opener of the invention. The mentioned person may take the many examples of fusions of DNA binding domains to other functional proteins as a guidance. It is for instance entirely possible to modify to hybrid systems such that upon association of the two parts of the hybrid system, an opener of the present invention is generated. In a preferred embodiment said opener is a fusion protein comprising at least a functional part of a mentioned opener, and a sequence specific nucleic acid binding domain. Preferably the opener comprises at least a functional part of a histone-acetyltransferase, a histone methyltransferase or a chromatin-remodelling protein. Preferably, said histone-acetyltransferase comprises a p300/CBP protein, a P/CAF protein, or a CBP protein or a functional part, derivative and/or analogue thereof. Preferably, said histone methyltransferase comprises an Ashl protein or a functional part, derivative and/or analogue thereof. Said chromatin- remodelling protein preferably comprises a trithorax group protein, a CHRAC group protein, a NURF group protein, ACF group proteins or a functional part, derivative and/or analogue thereof. The mentioned openers may be fused to the DNA specific binding domain of a zinc-finger protein, a bacterial DNA binding protein, a yeast or fungus DNA binding protein. Preferably, said DNA binding protein is LexA or Gal4 or functional part, derivative and/or analogue thereof. Because openers or pre-openers have important activity in a cell it is important not to express to much of these proteins in the cell hnes mentioned as this can have toxic effects. This toxic effect is significantly less in cases where a pre-opener has been converted into an opener by providing it with a sequence specific binding activity. This localizes the effects somewhat, although titration effects may still occur also in this situation. Dosed expression is therefore also of importance for cell lines, provided with an expression unit for an (pre-)opener provided with a sequence specific binding activity. The DNA binding domain is typically added to the N-terminal or the C-terminal end of a protein of the invention. On occasion one of these fusion may not be functional, however, typically at least one of such chimera retains both properties of the fusion partners (domains). In case a fusion is intended with a protein comprising a SET-domain it is sometimes better to fuse the DNA binding domain at the side of the chain that is the furthest away from the SET domain. However, this is not a general rule.

Protein expression units may be provided with desirable features to implement certain desired functionality. For instance, enhancers, introns, suitable untranslated regions etc. may be used. Inducible promoters or constitutive promoters may be used. Thus the present invention further provides a method, wherein the expression unit for the protein of interest and/or an expression unit expressing an opener of the invention is provided with an additional transcription/translation regulating and/or stimulating sequence. A method of the invention provides a high predictable expression. It also provides a high expression level. Moreover it also provides stable expression levels. In transfections of protein expression units it further provides more different integration events that (i) express the protein of interest at a high level, and results in (ii) a higher number of colonies that have expression of the protein of interest and (iii) in more colonies that have a suitable expression level for protein production. Both properties, of course, are compared to transfection with the same expression unit in the absence of the binding site for a member of a chromatin modification system of the invention. In a particularly preferred embodiment a protein expression unit is provided with a locus control region or a part thereof. Examples of such sequences may be found in sequences from the α or β globin locus as described US 5,610,053, WO96/04390 or the Igf2-locus. Of course, so-called UCOE sequences as described in WO 00/05393 and WO 02/24930 may also be used. In a preferred embodiment a nucleic acid of the invention further comprises a so-called STabilizing and Anti-Repressor sequence also termed STAR sequence. Examples of suitable STAR sequences are given in table 1. Other STAR sequences can be obtained from PCT/NL02/00390 filed in the name of

Chromagenics B.V, which is incorporated by reference herein. This filing also contains methods for finding other STAR sequences. Such other STAR sequences are of course also capable of being used in the present invention. STAR sequences can confer upon a linked expression cassette improved transcription capabilities, including but not limited to at least partial protection from repression stimulating influences of DNA adjacent to integrated foreign nucleic acid. Placement of the signal sequences and elements on the nucleic acid to be transfected depends on the particular signal or element. A STAR sequence is preferably placed outside of an expression cassette. Preferably an expression cassette is flanked by at least two STAR sequences. A STAR sequence at least in part improves the predictability of expression of a transferred nucleic acid leading to a larger proportion of cells having a suitable expression pattern. This is especially so for embodiments wherein two or more expression cassettes are transferred of which expression is desired. The presence of a STAR sequence, preferably on each of the thus transferred cassettes improves the number of cells selected with a method of the invention. As mentioned above, an increase in the number of cells with appropriate expression patterns is very important in the selection of production cells for clinical grade polypeptides. Thus in one embodiment of the invention said protein expression unit comprising said nucleic acid binding site for said opener is flanked on one or both sides by a nucleic acid comprising a STabilizing Anti-Repressor (STAR) sequence. The binding site for said opener is of course preferably placed together with the transcription unit on the side of the STAR sequence. A STAR element further improves stability, levels and predictability of expression of the protein expression unit. It further significantly increases the number of clones expressing a high amount of protein. Without being bound by theory it is believed that STAR elements create so-called nucleic acid domains of common regulation, if only to prevent the action of transcription repressors located outside the domain from influencing the domain. By placing a binding site for an opener present in the cell, in the domain that domain is preferentially opened and actively maintained in an open state. Optimal results are obtained when the binding site is operably linked to a promoter present said expression unit. By operably linkage is meant that a bound opener is capable of affecting accessibility of the chromatin comprising said promoter. Preferably, the binding site is provided to the expression unit upstream of a promoter therein. Typically though not necessarily good results are obtained when the binding site is within 10 bases from the promoter together with promoter associated factor binding sites, preferably, upstream of said promoter. (It is of course possible to introduce further binding sites for openers in or the vicinity of the protein expression unit. Such additional binding sites may further improve expression characteristics. All mentioned advantages of the invention are of course compared to the same protein expression unit but lacking either the binding site and/ or the additional element such as the STAR sequence. A further aspect of the invention is the combination in the protein expression unit of an opener in the context of the invention with a TRAnscription Pause (TRAP) sequence. This combination further improves the predictability of expression of the protein of interest. The invention uses the mentioned combination to enhance a protein expression characteristic of a protein expression unit. It is thought that a TRAP at least in part prevents the formation of antisense RNA or at least in part prevents transcription to enter said protein expression unit. TRAP sequences are described in PCT/NL03/00850 filed in the name of Chromagenics and this reference is therefore incorporated by reference herein for definition of TRAP sequences and for methods for providing protein expression units with TRAP sequences. Usually DNA sequences such as the SV40 polyadenylation signal are used to terminate transcription by placing the SV40 polyadenylation signal immediately downstream of a gene that is expressed. In other words, transcription should be prevented from continuing downstream of the gene. In the present invention transcription blockers (TRAP) are preferably placed both upstream and downstream of the expression unit, in such a manner that they prevent transcription to enter an open reading frame (when the TRAP is downstream thereof) or to enter the combination of the promoter and open reading frame driven by it (when located upstream of the open reading frame). The orientation of TRAP when placed downstream is opposite of the usual orientation of the SV40 polyadenylation signals that are placed downstream of genes. The orientation of an upstream TRAP is in the same orientation as the SV40 polyadenylation signals that are placed downstream of the genes. In one embodiment a method of the invention further comprises providing said cell with at least one protein expression unit which unit comprises a promoter functionally linked to an open reading frame encoding said at least one protein of interest, characterised in that said protein expression unit further comprises at least one TRAnscription Pause (TRAP) sequence and wherein said TRAP sequence is functionally located downstream of said open reading frame and at least in part prevents formation of antisense RNA. Preferably, said at least one TRAP sequence is in a 3'-5' orientation (in relation to said coding region).

Preferably, said TRAP sequence reduces the formation of antisense RNA to a non-detectable level. Due to the presence of said TRAP the formation of antisense RNA is at least in part prevented and hence the amount of dsRNA is decreased. As a consequence, the level of small dsRNAs of 21 to 23 basepairs (RNAi) is also decreased and the corresponding (full length) RNA encoding a protein of interest will not be degraded. Hence, translation of said corresponding RNA results in (increased) expression of a protein of interest. Surprisingly, as disclosed herein with the experimental part the use of

TRAP sequences further improves stability of expression of the protein of interest in the protein expression unit.

In the above -outlined embodiment, the TRAP sequence can for example be a terminator and/or a polyadenylation signal sequence, but in an orientation which differs from a possibly used terminator sequence behind an open reading frame in said protein expression unit. However, it is entirely possible that there are TRAP sequences that are bi-directional. These may also be used in the present invention to at least in part prevent transcription from entering a transcription unit.

Further provided is use of a TRAP to strengthen 'inertness' of the transcription units for instance when the CMV-driven transcription in the units of FIGl A are also prevented from escaping the transcription unit. Normally the SV40 transcriptional terminator is used for this purpose. This terminator does not, however, stop transcription completely. Hence a further TRAP sequence is incorporated upstream of the 3' STAR element in the expression cassette (FIG. 2C). This TRAP sequence is placed in a 5'- 3' orientation, in order to stop transcription that might leak through the SV40 transcriptional terminator. In this configuration the entire expression cassette has become essentially inert for transcription leaking in as well as leaking out. Thus in another embodiment the invention provides the use of a TRAP sequence to at least in part isolate a genetic element from transcription proceeding into the element. In a preferred embodiment the genetic element is a STAR element. Thus the invention further provides a STAR element together with a TRAP sequence of the invention. Preferably, a STAR element flanked by at least two STAR elements on either side. The orientation of the TRAP element in these embodiments is such that transcription proceeding from outside the STAR element into the STAR element is at least in part prevented. This embodiment is in particular relevant if there were to be inverted repeats present in the STAR element. These inverted repeats can initiate the formation of dsRNA. This in turn would lead to gene silencing of adjacent genes. Thus, this specific configuration of TRAP -STAR-TRAP elements can not only prevent formation of dsRNA in the genetic element, i.e., the STAR element, it also provides further protection of the entire expression unit.

In another embodiment, the invention provides a method for expression (or producing) of at least one protein of interest in a cell comprising providing said cell with at least one protein expression unit which unit comprises a promoter functionally hnked to an open reading frame encoding said at least one protein of interest, characterised in that said protein expression unit further comprises at least one TRAP sequence and wherein said TRAP sequence is located upstream of said promoter and at least in part prevents transcription to enter said protein expression unit. Preferably, said at least one TRAP sequence is in a 5'-3' orientation (in relation to said coding region). Again, a TRAP sequence used in the latter embodiment can be a terminator and/or a polyadenylation signal sequence, but this time the TRAP sequence is in an unusual position with regard to the open reading frame, because said TRAP is located upstream of the promoter that drives expression of said open reading frame.

In this embodiment, the presence of a TRAP sequence at least in part prevents transcription from a promoter sequence located outside a protein expression unit. Hence, the RNA from the protein expression unit does not have to compete with other RNA and hence a more efficient protein production system is provided.

The use of a TRAP to at least in part prevent formation of antisense RNA or to at least in part prevent transcription to enter said protein expression unit isolates said protein expression unit from negative effects, like formation of RNAi, from outside said unit.

A TRAP sequence is herein functionally defined as a sequence capable of at least in part prevent formation of antisense RNA or to at least in part prevent transcription to enter said protein expression unit. In other words a TRAP sequence, when placed into a transcription unit, results in a reduced level of transcription of the nucleic acid present on the 3'-side of the TRAP when compared to the level of transcription observed in the nucleic acid on the 5^!-side of the TRAP. When in this application no particular reference is made toward the orientation of the TRAP in a particular construct, it is in the orientation that it blocks transcription from entering a (potential) transcription unit, i.e. the transcription unit of the nucleic acid of interest. Preferably, the TRAP sequence is physically hnked to the protein expression unit that it aims to transcriptionally isolate from any flanking transcription units, at least prior to transfecting the unit into the genome of the cell. Upon integration of the unit, the unit and elements linked thereto become linked to sequences in the genome and the element present therein, in the case of concatemer integration the integrated unit can become hnked to co-integrated units or other transfected nucleic acid. In these embodiments a TRAP can be present upstream or downstream of the transcription unit it aims to isolate. When it is present upstream, the orientation of the TRAP is such that it can at least in part reduce transcription originating upstream of the transcription unit and the TRAP and proceeding toward the transcription unit. When it is present downstream of the transcription unit the TRAP is, in these embodiments, in an orientation that it at least in part reduces transcription origination downstream from the transcription unit that it is Hnked to and proceeding toward the transcription unit. The orientations upstream or downstream are typicaUy mirror images of each other. However, as mentioned above, in the situation where concatmers are formed upon integration of a protein expression unit in the genome, it is also possible to prevent transcription from entering a flanking co-integrated transcription unit by placement of a TRAP sequence downstream of the protein expression unit in the orientation that it reduces transcription initiating within the protein expression unit. In this embodiment, the TRAP is, prior to integration physicaUy linked to the transcription unit of which transcription can enter a flanking transcription unit. Through the hnkage of the TRAP to the unit prior to integration, this potential is at least in part reduced. This TRAP sequence is in addition to normal the transcription termination and/or a polyadenylation signals present a protein expression unit. With respect to the placing of a TRAP in relation to the protein expression unit it intends to protect from incoming transcription it is understood that the TRAP is preferably placed close to the expression cassette that it intends to transcriptionaUy isolate. In other words it is preferred that there are no potentially active promoter elements firing into the protein coding domain between the TRAP and the protein coding domain of the expression unit it intends to transcriptionally isolate, other than the promoter designed to direct transcription in the transcription unit (i.e. necessary to drive the protein of interest). As disclosed herein within the experimental part, a TRAP sequence can for example be a polyadenylation site and/or a pausing site, where the RNA polymerase II staUs. A TRAP can be derived from any source, as long as efficient termination of transcription is achieved. In one embodiment a TRAP is identified based on its ability to at least in part prevent formation of antisense RNA or to at least in part prevent transcription to enter said protein expression unit. Example 1 provides a method to test the effect of putative TRAPs on transcription. It is shown that STAR elements 7, 17 and 40 are poor in blocking transcription On the other hand, certain regions of phage λ, intergenic regions that separate histone H3 genes, as weU as a synthetic polyA sequence fulfil the criteria of a TRAP, since they are aU potent blocker of transcription.

In a preferred embodiment, said at least one TRAP sequence is located upstream of said promoter and wherein said TRAP sequence is in a 5'-3' orientation. In yet another preferred embodiment, said at least one TRAP sequence is located downstream of said open reading frame and wherein said TRAP sequence is in a 3'-5' orientation with respect to the orientation of the open reading frame. It is clear from the examples disclosed herein that the potential of TRAP sequences is orientation-dependent. It is therefore clear that the orientation in which a TRAP is applied to flank a transgene, can be of importance for its proper functioning. However, it is clear that there are also TRAP sequences which act independent of their orientation.

In a preferred embodiment, said protein expression unit comprises at least two TRAP sequences. A particularly preferred version of the at least two TRAP embodiment is the presence of at least one TRAP upstream and at least one TRAP downstream of the transcription unit of interest. Thus preferably, said at least two TRAP sequences are arranged such that said TRAP sequences are flanking the combination formed by said promoter and said open reading frame. When multiple protein expression units are present on one and the same part of genetic information it is also possible to at least partly inhibit or block transcription from one protein expression unit into another protein expression unit. In this case a TRAP sequence is placed between (possible different) protein expression units, the orientation of this TRAP sequence is of course in the 5'-3' orientation with respect to transcription for which the blocking is intended. When two expression cassettes integrate in a convergent manner transcriptional inert domains can be created by placing TRAP sequences in such a configuration that transcription is prevented to enter the transcription units.

Preferred examples of TRAP sequences are outHned in table 2. Preferably, said TRAP sequence comprises the lambda 35711-38103 sequence as depicted in table 2 and/or a functional equivalent and/or a functional fragment thereof. In another preferred embodiment, said TRAP sequence comprises a polyA sequence preferably a synthetic polyA (SPA) sequence and/or a functional equivalent and/or a functional fragment thereof, for example a SPA sequence and/or a functional equivalent and or a functional fragment thereof as depicted in table 2. In yet another preferred embodiment, aid TRAP sequence comprises a combination of an SPA and the human α2 globin gene pause signal and/or a functional equivalent and/or a functional fragment thereof, for example a combination of a SPA and the human α2 globin gene pause signal and/or a functional equivalent and/or a functional fragment as depicted in table 2.

A functional equivalent and or a functional fragment of a sequence depicted in 2 is defined herein as foUows. A functional equivalent of a sequence as depicted in table 2 is a sequence derived with the information given in table 2. For instance, a sequence that can be derived from a sequence in table 2 deleting, modifying and/or inserting bases in or from a sequence listed in table 2, wherein said derived sequence comprises the same activity in kind, not necessarily in amount, of a sequence as depicted in table 2. A functional equivalent is further a sequence comprising a part from two or more sequence depicted in table 2. A functional fragment of a sequence in table 2 can for example be obtained be deletions from the 5¹ end or the 3' end or from inside of said sequences or any combination thereof, wherein said derived sequence comprises the same activity in kind, not necessarily in amount.

Methods of the invention provide improved predictability, levels (yield) and stability of transgene expression. STAR elements increase the predictability, yield and stability of transgene expression even further by keeping chromatin- associated repression "out". Beside protection against chromatin-silencing by means of STAR elements, the present invention in addition creates means and methods to convert the chromatin of a transgene in a more open state, thus further facihtating the predictability, yield and stability of transgenic protein expression. To achieve this goal, chromatin- remodelHng proteins, histone acetyltransferase or histone methyltransferase proteins can be targeted to the promoter of the transgene. The invention thus prevents silencing of transgene expression by the combined action of keeping repression out and by simultaneously keeping chromatin in an open state. By combining STAR elements and or TRAP sequences with chromatin opening factors the present invention employs two or more different types of DNA elements or proteins that synergisticaUy reinforce each other to create novel (host) cells/cell lines that efficiently and stably express proteins. In a preferred embodiment openers, STAR elements and TRAP sequences as disclosed herein are combined in expression units. In combination with STAR elements, TRAP sequences potentiate the action of STAR elements. That is, incorporation of the STAR-TRAP combination results in higher expression levels than when STAR or TRAP elements alone are incorporated.

In principle any type of polypeptide or protein may be produced using a method of the invention. The method is particularly suited for the production of multimeric proteins comprising said at least to polypeptides. In a preferred a method provides for the expression of said at least two polypeptides in a predetermined ratio. Preferably, said at least two polypeptides comprise an immunoglobuHn heavy chain and an immunoglobuHn light chain. According to this embodiment a multimeric protein, an antibody, is obtained. It is clear to a person skilled in the art that it is possible to provide a ceU which expresses an immunoglobuHn heavy chain from one protein expression unit and an immunoglobulin hght chain from another protein expression unit with a third protein expression unit encoding a secretory component or a joining chain. In this way the production of for example slgA and pentameric IgM is provided. Preferably, said first polypeptide and said second polypeptide comprise at least the variable part of an immunoglobuHn hght chain and immunoglobuHn heavy chain. Preferably said first polypeptide comprises at least the variable part of an immunoglobulin heavy chain, whereas said second polypeptide comprises an immunoglobuHn light chain or derivative and/or analogue thereof. This embodiments warrants that an increased proportion of the ceUs selected will display a tendency to slightly overproduce immunoglobuHn heavy chain thereby aUowing more efficient production of the multimeric protein. ImmunoglobuHn technology is very advanced at the present time and it is possible to generate coding domains for antibodies that have no complementary antibody in nature, i.e. a completely artificial antibody. Such antibodies are also within the scope of the present invention. For an overview of relevant technology for antibodies, their selection and production we refer to (Chad, HE, and Chamow, SM, 2001, Therapeutic antibody expression technology Curr Opin Biotechn 12, 188-194. Das, RC, 2001. Proteins and antibodies make advances as therapeutic products. Am CHn Lab 20, 8-14.) Further provided is the use of a ceU of the invention, for the production of an antibody or a functional part, derivative and or analogue thereof. In a particularly preferred embodiment the antibody produced comprises a human or humanized antibody, whereas the ceU wherein the antibody is produced is a human ceU or derived there from, for instance by fusion of a human ceU to a human ceU or a non-human ceU. In view of the production of multimeric protein a method of the invention preferably further comprises providing said ceU with a second protein expression unit. Preferably, said expression unit encodes a member of a multimeric protein. Preferably, said protein expression unit encodes an immunoglobuHn heavy or Hght chain or a antigen binding part, derivative and/or analogue thereof.

A method of the invention is particularly suited for the selection of ceUs for production of clinical grade polypeptide of interest. A method therefore preferably, further comprises culturing said cell and harvesting said (multimeric) protein. The invention therefore further provides a sample comprising said protein, obtainable by a method of the invention. Preferably a sample comprising at least the variable part of an immunoglobuHn Hght chain and immunoglobuHn heavy chain. Preferably, wherein said protein comprises a human immunoglobuHn light chain and a human immunoglobuHn heavy chain or immunologically related to a human immunoglobulin chain. The invention further provides the use of a sample or antibody of the invention for the preparation of a medicament or a vaccine. Preferably for the treatment of cancer.

The protein expression unit may be mon-cistronic, bi-cistronic or multicistronic. Preferably, said protein expression unit comprises a multicistronic gene. Units comprising several cistrons can be transcribed as a single mRNA. Translation of the second and further coding regions present on that RNA can be achieved in various ways, including the use of translation reinitiation sites or internal ribosome entry sites, the latter of which is preferred. Advantages of bi- or multi-cistronic units are plurifold and include easy selection of clones expressing a protein of interest, for instance by placing the nucleic acid encoding a dominant selectable marker protein downstream of nucleic acid encoding a protein or polypeptide of interest.

Any type of promoter may be used in the present invention as long as it is operable for aUowing transcription in the protein expression unit at a certain time point, or continuously. Preferred promoters encompass the a human cytomegalovirus, a simian virus 40, a ubiquitine C, an elongation factor one-alpha promoter or a functional part, derivative, analogue or combination thereof. Functional part can be generated by deletion or mutation of nucleic acid of the promoter. A derivative is for instance a promoter of a different species but homologous to a promoter mentioned above. Such promoters can be found amongst others by comparing sequences of the different species. Human cytomegalovirus has a homologue in other species, similarly simian virus 40 has a homologue in other species. Promoters found in such homologues are also preferred in the present invention. Analogues of such promoters are promoters comprising one or more similar elements found in the mentioned promoters but obtained artificiaUy or from a different promoter. Such elements may comprise a certain transcription initiation region (TATAA box or equivalent such as the promoter driving hADA.). Further elements are particular enhancing elements placed in the vicinity of the transcription initiation region and the Hke.

The invention further provides a ceU obtainable by a method of the invention. Said cell of course comprising an expression unit comprising a binding site for an opener of the invention. Preferably, said expression unit is provided with a binding site for said opener. Further provided is a cell provided with a nucleic acid encoding an opener of the invention. Preferably, an opener provided with a new binding DNA binding specificity. Preferably, said ceU is a yeast ceU, a vertebrate cell or a plant ceU. Preferably mammalian ceUs and of these preferably a human ceU. Of course methods of the invention may be performed in vitro or in vivo. Preferably, however, a method of the invention is preformed in vitro. Preferred ceU Hnes are cell Hnes used for the production of proteins. Of these, a preferred ceU is a U_ 2 OS osteosarcoma, CHO, 293, HuNS-1 myeloma, WERI-Rb-1 retinoblastoma, BHK, Vero, non- secreting mouse myeloma Sp2/0-Ag 14, non-secreting mouse myeloma NSO, or NCI-H295R adrenal gland carcinoma ceU. The invention further provides a nucleic acid comprising a protein expression unit which is provided with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener), preferably, further comprising a STAR sequence. Preferably said expression unit comprises a human cytomegalovirus, a simian virus 40, a ubiquitine C, an elongation factor one-alpha promoter or a functional part, derivative, analogue or combination thereof.

The invention further comprises a the use of an opener for stabihzing expression of an expression unit and the use of an opener for increasing the number of clones expressing a certain amount of protein after genetic modification. Also provided is the use of an opener for increasing transcript levels produced by an expression unit.

In yet another embodiment the invention provides a method for improving a function of a protein expression unit comprising a STAR element comprising providing said protein expression unit with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener). Also provided is a method for improving a function of a STAR element in a protein expression unit comprising providing said protein expression unit with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener). The targeting of chromatin openers to a transgene or a promoter of a transgene is used to achieve predictable, high yields and stable transcription of a transgene. In the present invention HAT proteins such as p300, CBP, and/or P/CAF, HMTase proteins such as Ashl or the Brahma protein or functional relevant parts of these proteins are produced as fusion protein with the LexA protein (FIG 1A,B) (Bunker and Kingston 1994).

Instead of LexA-Opener fusion proteins that targeted to LexA binding sites, also GAL4-Opener fusion proteins can be used. These GAL4-Opener fusion proteins are targeted to GAL4 binding sites, which are placed upstream of a promoter. Unlike the bacterial LexA protein, GAL4 is a yeast protein. Like LexA protein, GAL4 is a transcription factor that has a DNA binding domain and a trans-acting domain, the last domain being responsible for activation of gene expression. To create a GAL4-Opener fusion protein, the part of the GAL4 gene, encoding amino acids 1 to 147 (Lilhe and Green, 1989), is cloned in frame with the respective Opener protein or functional part of the Opener protein. In the current invention expression of the GAL4-Opener fusion gene is driven by the SV40 promoter. The GAL4-Opener fusion protein is targeted to GAL4 binding sites, called GAL4 operators. Commonly four GAL4 operators are placed immediately upstream of a promoter. One GAL4 operator is the following sequence: CGGAGTACTGTCCTCCG.

These fusion proteins are placed under control of an inducible or constitutive promoter such as the SV40 promoter (FIG1 A,B) The expression unit for these fusion proteins are present on the same plasmid as the expression unit that contains the gene that encode the protein of interest (Gene 1) (FIG 1). Gene 1 is placed under control of the CMV promoter. Upstream of the CMV promoter binding sites are cloned to which the LexA- HAT, LexA-HMTase or LexA-Brahma proteins are targeted (FIG 1A). Thus these fusion proteins are targeted to the vicinity of the promoter to keep open the chromatin structure of the promoter in order to faciHtate the assessabihty of the promoter for transcription factors. It is also possible to create one plasmid containing three expression units that encode respectively Gene 1, Gene 2 and LexA-HAT, LexA-HMTase or LexA-Brahma (FIG IB). The expression units encoding Gene 1 and Gene 2 are oriented divergent in such a manner that the two CMV promoters are adjacent although differently oriented. Between the two promoters LexA binding sites are placed to which the LexA-fusion protein is targeted. In this manner chromatin openers s are targeted to both expression units.

It will also be clear to a person skilled in the art that it is not essential that the LexA fusion proteins or HAT proteins are expressed from the same plasmid that contains the expression unit with the gene of interest. The LexA fusion proteins or HAT proteins can also be produced from a separate plasmid.

Therefore, the invention provides in one embodiment, a method for obtaining a cell which expresses one or more proteins comprising providing said ceU with one or more protein expression units encoding said one or more proteins, characterised in that at least one but preferably at least two of said protein expression units comprises at least one chromatin opener and or one STAR sequence. In a preferred embodiment Gene 1 and Gene 2 encode the Hght and heavy chain of a multimeric immuno globin protein The terms "cell"/"host ceU" and "cell Hne'V'host cell line" are respectively typicaUy defined as a eukaryotic ceU and homogeneous populations thereof that are maintained in ceU culture by methods known in the art, and that have the ability to express heterologous or homologous proteins. Thus in the present invention it is possible to provide an opener to an expression unit present in the ceU, for instance by means of homologous recombination. Other features may also be provided to the expression unit in the cell. It is thus entirely possible to for instance activate a coding region present in the ceU. For instance the gene encoding erythropoietin is not normally expressed in a ceU. By introducing providing this protein expression unit with a binding site for an opener of the invention it is possible to obtain expression of the endogenous gene. This will typically also require replacement of the promoter or addition of further promoter elements such as enhancers. In this embodiment the endogenous gene is further preferably further provided with a STAR sequence on one side but preferably on both sides of the endogenous gene. Of course methods of the invention may also be used to enhance expression of already expressed endogenous genes. Means and methods for the activation or enhanced expression of endogenous genes by means of homologous or targeted recombination or known in the art. The present invention contributes binding sites for openers, STAR elements and particular preferred promoters to this technology.

The term "expression" is typicaUy used to refer to the production of a specific RNA product or products, or a specific protein or proteins, in a cell. In the case of RNA products, it refers to the process of transcription. In the case of protein products, it refers to the processes of transcription, translation and optionaUy post-translational modifications. In the case of secreted proteins, it refers to the processes of transcription, translation, and optionally post- translational modification (e.g. glycosylation, disfulfide bond formation, etc.), followed by secretion. In the case of multimeric proteins, it includes assembly of the multimeric structure from the polypeptide monomers. The corresponding verbs of the noun "expression" have an analogous meaning as said noun.

A protein or polypeptide is herein defined as being either (i) a product obtained by the processes of transcription and translation and possibly but not necessarily said product is part of a multimeric protein (for example a sub unit) and/or (n) a product obtained by the processes of transcription, translation and post-translational modification. The term "multimer" or "multimeric protein" is typicaUy defined as a protein that comprises two or more, possibly non- identical, polypeptide chains ("monomers"). The different monomers in a multimeric protein can be present in stoichiometricaUy equal or unequal numbers. In either case, the proportion of the monomers is usuaUy fixed by the functional structure of the multimeric protein.

The term "protein expression unit" is herein defined as a unit capable of providing protein expression and typicaUy comprises a functional promoter, an open reading frame encoding a protein of interest and a functional terminator, aU in operable configuration. A functional promoter is a promoter that is capable of initiating transcription in a particular ceU. Thus that is normaUy transcriptionally active in the cell that is used to obtain the expression of the protein of interest. With normaUy transcriptionally active is meant that the promoter must be capable of initiating transcription in the cell, which in case of an inducible promoter may encompass the provision of the inducer for the promoter. Thus so-caUed minimal promoters that have been stripped of their associated transcription initiating nucleic acid sequences are not encompassed in the term promoter as used in the present invention in the context of a promoter driving expression of the protein of interest. Examples of such minimal promoters are the SV40 minimal promoter (from Promega pGL3; Accession number U47296) and the LBK-AP minimal promoter (Ruez nsky, D., Beckman, H., & Kadesch, T. Modulation of the IgH enhancer's cell type specificity through a genetic switch. Genes Dev. 5, 29-37 (1991)).

Preferred promoters for obtaining expression in eukaryotic cells are the CMV-promoter, a mammahan EFl-alpha promoter, a mammalian ubiquitin promoter, or a SV40 promoter. A functional terminator is a terminator that is capable of providing transcription termination. One example of a suitable terminator is an SV40 terminator. The term "an open reading frame encoding a protein of interest (or a transgene)" is typically defined as a fragment of DNA which codes for a specific RNA product or products or a specific protein or proteins, and which is capable of becoming integrated into the genome of a host cell. It includes DNA elements required for proper transcription and translation of the coding region(s) of the transgene. Said DNA encoding said protein of interest/transgene can either be a DNA encoding a product obtained by the processes of transcription and translation (and possibly but not necessarUy said product is part of a multimeric protein, for example a subunit) or a product obtained by the processes of transcription, translation and post- translational modification.

The terms "recombinant cell/host cell" and "recombinant ceU Hne/host ceU line" are respectively typicaUy defined as a host ceU and homogeneous populations thereof into which a transgene has been introduced for the purpose of producing a heterologous protein or proteins. A STAR (STabilizing Anti-Repressor) sequence (or STAR element; the terms will be used interchangeably herein) is a DNA element that we have first identified in eukaryotic genomes on the basis of their abUity to block transgene repression. STAR-sequences can be identified (as disclosed for example in example 1 of EP 01202581.3) using a method of detecting, and optionally selecting, a DNA sequence with a gene transcription-modulating quaHty. A STAR sequence comprises the capacity to influence transcription of genes in cis and/or provide a stabifizing and/or an enhancing effect. The expression level of the transgene is stable over many cell generations, and does not manifest stochastic silencing. Therefore, STAR sequences confer a degree of position-independent expression on transgenes that is not possible with conventional transgenic systems. The position independence means that transgenes that are integrated in genomic locations that would result in transgene sUencing are, with the protection of STAR elements, maintained in a transcriptionaUy active state. Moreover, a STAR element is active in many different cell types.

Chromatin openers or simply openers (the terms will be used interchangeably herein) are involved in opening chromatin structure, through chromatin-remodelHng proteins and their complexes such as the Ashl protein, the Brahma protein, other trxG proteins or components of the CHRAC NURF and ACF group chromatin-remodeling complexes. Alternatively, chromatin openers are histone modifiers such as HAT proteins or functional relevant parts of such proteins that are stiU able to add acetyl groups to histone tails which has the consequence that the tight association between the basic histones and the acid DNA is loosened. Yet another class of Chromatin openers consists of specific histone methyltransferase such as the Ashl protein that add a methyl group to at least lysine 4 (K4) of histone H3 and that also results in opening of chromatin or making it more accessible to the general transcription machinery. Chromatin openers, these being chromatin- remodelling factors, specific HATs or HMTases or even other histone modifiers thus have in common that they faciHtate the binding of transcription factors to the promoter and hence increase the possibilities for transcription.

Transcription can be influenced through a direct effect of the regulatory element (or the protein(s) binding to it) on the transcription of a particular promoter. Transcription can however, also be influenced by an indirect effect, for instance because the regulatory element affects the function of one or more other regulatory elements. A gene transcription modulating quahty can also comprise a stable gene transcription quality. With stable is meant that the observed transcription level is not significantly changed over at least 5- 60 ceU divisions. A stable quality is useful in situations wherein expression characteristics should be predictable over many ceU divisions. Typical examples are cell lines transfected with foreign genes. Other examples are transgenic animals and plants and gene therapies. Very often, introduced expression cassettes function differently after increasing numbers of ceU divisions or plant or animal generations. Preferably, a stable quahty comprises a capacity to maintain gene transcription in subsequent generations of a transgenic plant or animal. Of course in case expression is inducible said quality comprises the quality to maintain inducibility of expression in subsequent generations of a transgenic plant or animal. Frequently, expression levels drop dramatically with increasing numbers of cell divisions. The present invention provides a means for at least in part counteracting this drop.

The present invention provides, amongst others, a method for obtaining a ceU which expresses one or more proteins. Openers are added to the expression unit of the gene of interest, preferably along with STAR elements that are appHed to flank the expression units, both chromatin openers and STAR elements being the basis of the stable expression of the transgene protein over many cell generations. We have demonstrated that STAR elements can protect individual transgenes from sUencing. Expression units that are not flanked by STAR elements can undergo significant silencing after only 5-60 culture passages, during which time sUencing of the STAR element protected units is neghgible.

The present invention uses chromatin openers and STAR sequences for the production of one or more proteins and thereby the invention provides (1) an increased predictability in the creation of recombinant cell Hnes that efficiently produce the heterologous multimeric proteins of interest, (2) an increased yield of the heterologous multimeric proteins, (3) stable expression of the heterologous multimeric proteins, even during prolonged cultivation in the absence of selection agent and (4) the invention also provides favorable transgene expression characteristics without amplification of the transgene. The increased yield of heterologous proteins provided by the invention may be obtained at low transgene copy numbers, without selective co-ampHfication using, for example, the DHFR/methotrexate system. This results in greater stabUity, since the transgene copy number is low and is not susceptible to decrease due to recombination (McBurney et al., 2002) or repeat-induced gene sUencing (Garrick et al., 1998). Fifth, the broad applicabiHty of the method of the invention includes its utUity in a wide range of host ceU lines. This is for example useful/desirable when a particular multimeric protein is preferably expressed by a particular host ceU line (e.g. expression of antibodies from lymphocyte-derived host ceU lines).

A method according to the invention therefore provides an improvement of expression of one or more proteins in a (host) ceU. In another embodiment the invention provides a method for identifying a ceU wherein expression of one or of more proteins in a predetermined ratio is comprising providing

- a coUection of cells with one or more protein expression units encoding said one or more proteins,

- selecting ceUs which express said one or more proteins, and - identifying from the obtained selection, ceUs that express said two or more proteins in said predetermined ratio, characterised in that at least two of said protein expression units comprise at least one chromatin opener

- identifying from the obtained selection, cells that express said two or more proteins in said predetermined ratio. Preferably, at least one of said expression units comprise at least one STAR sequence.

The selection of ceUs which express said one or more proteins can for example be obtained by performing a SDS-PAGE analysis, a Western blot analysis or an ELISA, which are aU techniques which are known by a person skUled in the art and therefore need no further elaboration. The identification of ceUs that express said two or more proteins in said predetermined ratio can also be performed by these techniques.

The presence of an opener and a STAR sequence in at least one of said protein expression units, again, provide the desired predictabUity, yield, stabUity of the one or more proteins. In another embodiment the invention provides a method wherein at least one of said protein expression units comprises a monocistronic gene comprising an open reading frame encoding a protein of interest and wherein said monocistronic gene is under control of a functional promoter. In yet another embodiment the invention provides a method according to the invention, wherein at least one of said protein expression units comprises a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein said bicistronic gene is under control of a functional promoter.

In a more preferred embodiment the invention provides a method according to the invention, wherein at least one of said protein expression units comprises

- a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein said bicistronic gene is under control of a functional promoter, which protein expression unit further comprises

- a monocistronic gene comprising an open reading frame encoding a second selection marker and wherein said monocistronic gene is under control of a functional promoter.

The term "bicistronic gene," is typically defined as a gene capable of providing a RNA molecule that encodes two proteins/polypeptides.

The term "monocistronic gene" is typically defined as a gene capable of providing a RNA molecule that encodes one protein/polypeptide.

The term "selection marker or selectable marker" is typicaUy used to refer to a gene and/or protein whose presence can be detected directly or indirectly in a ceU, for example a gene and or a protein that inactivates a selection agent and protects the host cell from the agent's lethal or growth- inhibitory effects (e.g. an antibiotic resistance gene and/or protein). Another possibility is that said selection marker induces fluorescence or a color deposit (e.g. green fluorescent protein and derivatives, luciferase, or alkaHne phosphatase). The term "selection agent" is typicaUy defined as a means for selecting for the presence of a selectable marker, such as an antibody. A dominant selection agent" is typically defined as a chemical compound that is able to kiU or retard the growth of host ceUs (e.g. an antibiotic). The term "dominant selection" is typically defined as the process of using a selection marker/selectable marker and a dominant selection agent to identify host ceUs with specific genetic properties (e.g. that the host ceU contains a transgene integrated into its genome).

The nouns "clone" and "isolate" typically refer to a recombinant host ceU Hne that has been identified and isolated by means of selection.

Improvements provided by a method according to the invention have at least three aspect which may be integrated or not. (1) With existing systems, recombinant ceU Hnes that simultaneously express acceptable quantities of the monomers of multimeric proteins can be created only at very low frequencies; the present invention increases the predictabUity of creating high-yielding recombinant host cell Hnes by a factor of ten or more. (2) Existing systems do not provide stoichiometricaUy balanced and proportional amounts of the subunits of multimeric proteins; the present invention ensures that the expression levels of the subunits will be balanced and proportional. (3) Existing systems do not provide a means of protecting the transgenes that encode the protein subunits from transgene sUencing.

FIG 1 provides a, non-Hmiting, schematic representation of one of the embodiments of this part of the invention. This is the configuration of the DNA elements of the expression units in the plasmid as well as after integration into the genome. Expression unit one is shown in FIG 1A. It contains an open reading frame for a transgene (a reporter gene, Genel). This is upstream of the attenuated EMCV IRES (Martinez-Sals et al 1999; Mizuguchi et al 2000; Rees et al 1996), and of the open reading frame encoding the zeocin resistance selectable marker protein (zeo). The gene cassette has the SV40 transcriptional terminator at their 3' ends (t). This bicistronic transgene is transcribed at high levels from the CMV promoter. Upstream of the CMV promoter are four LexA binding sites (LexA- BS). Next to this is the monocistronic gene encoding a fusion protein between the LexA protein and a histone acetyltransferase (HAT) or a functional part of a HAT that is stUl able to transfer acetyl groups to histone taUs (LexA-HAT). Alternatively a fusion protein is encoded between the LexA protein and a histone methyltransferase (HMTase) or a functional part of a HMTase protein that is stiU able to transfer a methyl group to at least lysine K4 of histone H3. Alternatively a fusion protein is encoded between LexA and the Brahma protein. Either one of these monocistronic transcription units is transcribed from the SV40 promoter. The genes have the SV40 transcriptional terminator at their 3' ends (t). This entire cassette with multiple genes is flanked by STAR elements. FIG IB is simUar as FIG 1A, but one plasmid contains now three expression units that encode respectively Gene 1, Gene 2 and LexA-HAT, LexA-HMTase or LexA-Brahma. The expression units encoding Gene 1 and Gene 2 are oriented divergent in such a manner that the two CMV promoters are adjacent although differently oriented. Between the two promoters LexA binding sites are placed to which the LexA fusion proteins are targeted. In this manner chromatin openers are targeted to both expression units.

It is clear to a person skUled in the art that in these examples more possible combinations can be made. For instance the expression units can be made in such a manner that Genel, Gene 2 and the LexA-HAT, LexA-HMTase or LexA-Brahma or HAT each are located on separate plasmids. Also STAR elements can be omitted from these constructs and stUl the expression of genel can be benefited form the presence of chromatin openers.

Selection of cells that harbour the depicted plasmids can be with co- transfection with for instance a plasmid that contains the puromycin resistance gene. A second selection step can then involve adding Zeocin to the culture medium, since the Zeocin resistance gene is coupled to the gene of interest (Gene 1). It is also possible to directly select on Zeocin, since the Zeocin resistance gene is coupled to the gene of interest (Gene 1) through an IRES sequence. It is also possible that the expression unit encoding the puromycin resistance gene is place on the same plasmid as depicted in FIG1. It is also clear to a person skiUed in the art that the possible combinations of selection markers is numerous. An example of a possible antibiotic is provided above. The one antibiotic that is particularly advantageous is zeocin, because the zeocin-resistance protein (zeocin-R) acts by binding the drug and rendering it harmless. Therefore it is easy to titrate the amount of drug that kills ceUs with low levels of zeocin-R expression, whUe aUowing the high-expressors to survive. All other antibiotic-resistance proteins in common use are enzymes, and thus act catalytically (not 1:1 with the drug). When a two-step selection is performed it is therefore advantageous to use an antibiotic resistance protein with this 1:1 binding mode of action. Hence, the antibiotic zeocin is a preferred selection marker. For convenience the zeocin antibiotic is in a two-step selection method combined with puromycin-R or hygromycin-R in the monocistronic gene. It is furthermore clear that it is also possible to combine an antibiotic selection marker with a selection marker which provides induction of fluorescence or which provide a colour deposit.

It is also clear to a person skiUed in the art that different promoters can be used as long as they are functional in the used ceU. The CMV promoter is considered the strongest avaUable, so it is preferably chosen for the bicistronic gene in order to obtain the highest possible product yield. Other examples of preferred promoters are house keeping gene promoters, preferred examples of such house keeping gene promoters are the mammalian promoters for EF1- alpha or ubiquitin. The good expression and stability of the SV40 promoter makes it well suited for expression of the monocistronic gene; enough selection marker protein (for example the antibiotic resistance protein puromycin-R in the example cited herein) is made to confer high expression of said selection marker. Hence, said SV40 promoter is preferentiaUy used as a promoter driving the expression of the selection marker.

In a preferred embodiment the invention provides a method wherein at least one of said protein expression units comprises at least two STAR sequences. In an even more preferred embodiment the invention provides a method wherein said protein expression unit comprising at least two STAR sequences is arranged such that said protein expression unit is flanked on either side by at least one STAR sequence. In yet an even more preferred embodiment the said at least two STAR sequences are essentially identical. Essentially identical STAR sequences are defined herein as STAR sequences which are identical in their important domains, but which may vary within their less important domains (the domains that confer the transcription stabUizing or enhancing quality), for example a point mutation, deletion or insertion at a less important position within the STAR sequence. PreferentiaUy said essentially identical STAR sequences provide equal amounts of transcription stabUizing or enhancing activity. The use of STARs to flank at least one protein expression unit is one of the aspects of the balanced and proportional levels of expression of two or more proteins and more specificaUy for the expression of the monomers of multimeric proteins. The STARs create chromatin domains of definite and stable transcriptional potential. As a result, promoters that drive transcription of each bicistronic mRNA wiU function at definite, stable levels. A recombinant host ceU line created by the method of the invention is readily identified in which these levels result in appropriate proportions of each monomer of the multimeric protein of interest being expressed at high yields.

Yet another preferred feature of a method according to the invention is the introduction of a (weak) Internal Ribosome Binding Site (IRES) as an example of a protein translation initiation site with a reduced translation efficiency, between the open reading frame of the protein of interest and the selection marker open reading frame. Translation of proteins from IRES elements is less efficient than cap-dependent translation: the amount of protein from IRES-dependent open reading frames (ORFs) ranges from less than 20% to 50% of the amount from the first ORF (Mizuguchi et al., 2000). This renders IRES elements undesirable for production of aU subunits of a multimeric protein from one messenger RNA (mRNA), since it is not possible to achieve balanced and proportional expression of two or more protein monomers from a bicistronic or multicistronic mRNA. However, the reduced efficiency of IRES-dependent translation provides an advantage that is exploited by the current invention. Furthermore, mutation of IRES elements can attenuate their activity, and lower the expression from the IRES- dependent ORFs to below 10% of the first ORF (Lopez de Quinto & Martinez- Salas, 1998, Rees et al., 1996). The advantage exploited by the invention is as follows: when the IRES-dependent ORF encodes a selectable marker protein, its low relative level of translation means that high absolute levels of transcription must occur in order for the recombinant host cell to be selected. Therefore, selected recombinant host cell isolates wUl by necessity express high amounts of the transgene mRNA. Since the recombinant protein is translated from the cap -dependent ORF, it can be produced in abundance resulting in high product yields.

It is clear to a person skUled in the art that changes to the IRES can be made without altering the essence of the function of the IRES (hence, providing a protein translation initiation site with a reduced translation efficiency), resulting in a modified IRES. Use of a modified IRES which is stiU capable of providing a small percentage of translation (compared to a 5' cap translation) is therefore also included in this invention.

In yet another embodiment the invention provides a method for obtaining a cell which expresses two or more proteins or a method for identifying a ceU wherein expression of two or more proteins is in a predetermined ratio, wherein each of said protein expression units resides on a separate DNA-carrier. The present invention preferentiaUy makes use of a separate transcription unit for each protein and/or monomer of a multimeric protein. In each transcription unit the monomer ORF is produced by efficient cap-dependent translation. This feature of the invention contributes that recombinant host ceUs are isolated which have high yields of each monomer, at levels that are balanced and proportionate to the stoichiometry of the multimeric protein. The increased predictabUity at which such recombinant host ceUs are isolated results in an improvement in the efficiency of screening for such isolates by a factor often or more. In a preferred embodiment said DNA-carrier is a vector (or plasmid; the terms are used interchangeably herein). In another embodiment said vector is a viral vector and in a more preferred embodiment said viral vector is an adenoviral vector or a retroviral vector. It is clear to person skiUed in the art that other viral vectors can also be used in a method according to the invention.

Conventional expression systems are DNA molecules in the form of a recombinant plasmid or a recombinant viral genome. The plasmid or the viral genome is introduced into (mammalian host) ceUs and integrated into their genomes by methods known in the art. The present invention also uses these types of DNA molecules to deliver its improved transgene expression system. A preferred embodiment of the invention is the use of plasmid DNA for dehvery of the expression system. A plasmid contains a number of components: conventional components, known in the art, are an origin of rephcation and a selectable marker for propagation of the plasmid in bacterial cells; a selectable marker that functions in eukaryotic cells to identify and isolate host cells that carry an integrated transgene expression system; the protein of interest, whose high-level transcription is brought about by a promoter that is functional in eukaryotic ceUs (e.g. the human cytomegalovirus major immediate early promoter/enhancer, pCMV (Boshart et al., 1985)); and viral transcriptional terminators (e.g. the SV40 polyadenylation site (Kaufman & Sharp, 1982)) for the transgene of interest and the selectable marker.

The vector used can be any vector that is suitable for cloning DNA and that can be used in a transcription system. When host ceUs are used it is preferred that the vector is either an integrating vector or an episomaUy repHcating vector. In an episomaUy rephcating vector, effects due to different sites of integration of the vector are avoided. DNA elements flanking the vector at the site of integration can have effects on the level of transcription of the promoter and thereby mimic effects of fragments comprising DNA sequences with a gene transcription modulating quality. In a preferred embodiment said vector comprises a rephcation origin from the Epstein-Barr virus (EBV), OriP, and a nuclear antigen (EBNA-1). Such vectors are capable of repHcating in many types of eukaryotic ceUs and assemble into chromatin under appropriate conditions. In a most preferred embodiment the invention provides a method for obtaining a cell which expresses two or more proteins or a method for obtaining a cell wherein expression of two or more proteins is in a predetermined ratio comprising providing two or more protein expression units wherein one of the said protein expression units or said protein(s) of interest encodes an immunoglobuHn heavy chain and/or wherein another of the said protein expression units or said protein(s) of interest encodes an immunoglobuHn light chain. According to this embodiment a multimeric protein, an antibody, is obtained. It is clear to a person skiUed in the art that it is possible to provide a cell which expresses an immunoglobulin heavy chain from one protein expression unit and an immunoglobulin light chain from another protein expression unit with a third protein expression unit encoding a secretory component or a joining chain. In this way the production of for example slgA and pentameric IgM is provided. In yet another embodiment a method according to the invention is provided, wherein said protein expression units are introduced simultaneously into said cell.

Preferably, a functional promoter is a human cytomegalovirus (CMV) promotor, a simian virus (SV40) promoter, a human ubiquitin C promoter or a human elongation factor alpha (EFl-α) promoter.

In another embodiment the invention provides a protein expression unit comprising

- a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein said bicistronic gene is under control of a functional promoter and

- at least one chromatin opener and

- at least one STAR sequence and/or - at least two TRAP sequences.

In a more preferred embodiment said protein expression unit comprises a chromatin opener and at least two STAR sequences which are preferentially arranged such that said protein expression unit is flanked on either side by at least one STAR sequence. Examples of such a protein expression unit are provided within the experimental part of this patent appHcation.

In another embodiment the protein expression unit according to the invention comprises STAR sequences, wherein said STAR sequences are essentiaUy identical. In a preferred embodiment the invention provides a protein expression unit comprising

- a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein said bicistronic gene is under control of a functional promoter - at least one STAR sequence, and is optionaUy provided with a monocistronic gene cassette, wherein said STAR sequence is depicted in Table 1 and/or a functional equivalent and/or a functional fragment thereof. - at least two TRAP sequences that are positioned to flank the STAR elements and wherein said TRAP sequences are depicted in Table 2. In another embodiment a protein expression unit according to the invention is provided wherein said protein translation initiation site with a reduced translation efficiency comprises an Internal Ribosome Entry Site (IRES). More preferably a modified, e.g. weaker, IRES is used.

In yet another embodiment a protein expression unit according to the invention is provided wherein said protein expression unit is a vector. In a preferred embodiment said DNA-carrier is a vector (or plasmid; the terms are used interchangeably herein). In another embodiment said vector is a viral vector and in a more preferred embodiment said viral vector is an adenoviral vector or a retroviral vector. It is clear to person skiUed in the art that other viral vectors can also be used in a method according to the invention.

In a preferred embodiment a protein expression unit according to the invention is provided, wherein said protein of interest is an immunoglobulin heavy chain. In yet another preferred embodiment a protein expression unit according to the invention is provided, wherein said protein of interest is an immunoglobulin light chain. When these two protein expression units are present within the same (host) cell a multimeric protein and more specifically an antibody is assembled. The invention includes a cell provided with a protein expression unit comprising a chromatin opener and a STAR element. The invention also includes a (host) ceU comprising at least one protein expression unit according to the invention. Such a (host) ceU is then for example used for large-scale production processes. The invention also includes a cell obtainable according to anyone of the methods as described herein. The invention furthermore includes a protein obtainable from said cell (for example, via the process of protein purification). Preferably, said protein is a multimeric protein and even more preferably said multimeric protein is an antibody. Such an antibody can be used in pharmaceutical and/or diagnostic apphcations.

The foregoing discussion and the following examples are provided for illustrative purposes, and they are not intended to Hmit the scope of the invention as claimed herein. They simply provide some of the preferred embodiments of the invention. Modifications and variations, which may occur to one of ordinary skill in the art, are within the intended scope of this invention. Various other embodiments apply to the present invention, including: other selectable marker genes; other IRES elements or means of attenuating IRES activity; other elements affecting transcription including promoters, enhancers, introns, terminators, and polyadenylation sites; other orders and/or orientations of the monocistronic and bicistronic genes; other anti-repressor elements or parts, derivations, and/or analogues thereof; other vector systems for delivery of the inventive DNA molecules into eukaryotic host ceUs; and apphcations of the inventive method to other transgenic systems.

EXAMPLES Example 1: Expression of LexA-HAT and LexA-Brahma and Brahma proteins in CHO cells

One object of this invention is to apply chromatin openers to improve the predictabUity, yield and stabUity of transgenes in mammahan ceU lines. Here we introduce several chromatin openers into CHO ceUs and we describe the construction of the various opener constructs.

Materials and Methods Plasmids

The construction of the pPlug&Play-d2EGFP-ires-Zeo (PP) vector is described below. Plasmid pd2EGFP (Clontech 6010-1) is modified by insertion of a Hnker at the BsiWl site to yield pd2EGFP-link. The linker (made by anneahng oligonucleotides GTACGGATATCAGATCTTTAATTAAG and GTACCTTAATTAAAGATCTGATAT) introduces sites for the Pad, Bglll, and EcoHV restriction endonucleases. This creates the multiple cloning site MCSII for insertion of STAR elements. Then primers (GATCAGATCTGGCGCGCCAT TTAAATCGTCTCGCGCGTTTCGGTGATGACGG) and (AGGCGGAT CCGAATGTATTTAGAAAAATAAACAAATAGGGG) are used to amplify a region of 0.37 kb from pd2EGFP, which is inserted into the Bglll site of pIRES (Clontech 6028-1) to yield pIRES-stuf. This introduces sites for the Ascl and Swal restriction endonucleases at MCSI, and acts as a "stuffer fragment" to avoid potential interference between STAR elements and adjacent promoters. pIRES-stuf is digested with Bglll and Fspl to Hberate a DNA fragment composed of the stuffer fragment, the CMV promoter, the IRES element (flanked by multiple cloning sites MCS A and MCS B), and the SV40 polyadenylation signal. This fragment is ligated with the vector backbone of pd2EGFP-link produced by digestion with BamHI and Stul, to yield pd2lRES- hnk. The open reading frames of the zeocin-resistance genes is inserted into the BamΗI/Notl sites of MCS B in pd2IRES-hnk as follows: the zeocin- resistance ORF is amplified by PCR with primers (GATCGGATCCTTC GAAATGGCCAAGTTGACCAGTGC) and (AGGCGCGGCCGCAATTCTCAG TCCTGCTCCTC) from plasmid pEM7/zeo, digested with BamTRl and Notl, and Hgated with .BαmHI/iVotl-digested pd2IRES-hnk to yield pd2IRES-link-zeo. The SEAP reporter ORF is introduced into pd2IRES-Hnk-zeo by PCR amplification of pSEAP2-basic with primers (GATCGAATTCTCGCGACTTCG CCCACCATGC) and (AGGCGAATTCACCGGTGTTTAAACTCATGTCTGCTC GAAGCGGCCGG), and insertion of the coRI-digested SEAP cassette into the EcoRl sites in MCS A of the plasmids pd2IRES-link-zeo (to yield plasmid PP2). PP2 is cut with EcoRI and Mlul to remove the SEAP gene and p2EGFP is introduced with primers (GATCGAATTCATGGTGAGCAAGGG CGAGGAG) and (AGGCACGCGTGTTAACCTACACATTGATCCTAGCA GAAGC). This vector is used as a basis vector to construct PP-LexA (PPL), PP-LexA-Brm

(PPLBrm),PP-LexA-PCAF (PPLPCAF), PP-LexA-p300HAT (PPLp300) and PP- LexA-AshlHMTase (PPLHuAshl ).

Brm coding sequence is pcr-amphfied from plasmid pSVhSNF-α (Chiba et al 1994) using primers Brm-alF-H3-AgeI (GATCAAGCTTACCGG TATGTCCACGCCCACAGACCCTGGTG C) and Brm-al572R-XbaI (AGGCTCTAGAATCACTCATCATCCGTCCCACTT CCTTC) and cloned into pPur (BD biosciences #6156-1) using Hindlll and Xbal to create pPur-Brm. LexA binding sites (LBS) are amplified from plasmid pREP4-HSF-Luc+ (van der Vlag et al, 2000) using primers LBS-for-Sall (AGGCGTCGACGTTTCG ACTCCCAAGCTTTG) and LBS-rev-AscI (GATCGGCGCGCCGGTACC ATAGCGGCCGCGAC) and cloned upstream of the CMV promoter in PP using Sail and Ascl to create PPLbs. LexA is amplified from plasmid pEG202 (Bennetzen and Hall, 1982) using primers LexA-for-H3 (GATCAAGCTTA TGAAGACGTTAACGGCCAGGC) and LexA-rev-Agel (AGGCACCGGTCAG CCAGTCGCCGTTGCGAATAACC) and cloned downstream of the SV40 promoter in plasmid pPur using Hindlll and Agel creating pPur-LexA. OHgo's Link-for-Bsu (GATCTCCCCTGAGGAAGTGC ACAACCTGAGGCC) and Link- rev-Bsu (GATCTGGCCTCAGGTTGTGCACT TCCTCAGGGG) are Hgated into the BamHI site of pPur-LexA to create pPur-LexA-Hnker.

The control vector PPlbs-lexA (PPL) is created by removing the puro coding sequence from pPur-LexA using Agel and Xbal foUowed by a transfer of the LexA cassette (ApaLI x EcoRI, blunted) into the EcoRV site of PPlbs The Brm pcr-product (primers Brm-alF-H3-AgeI and Brm-al572R-XbaI) is cloned into pPur-LexA using Agel and Xbal to create pPur-LexA-Brm. The P/CAF coding sequence is pcr-amplified from plasmid pCX-P/CAF (Martinez- Balbas et al, 2000) using primers PCAF-alF-h3-AgeI (GATCAAGCTTAC CGGTATGTCCGAGGCTGGCGGGGCCG) and PCAF-a833R-XbaI (AGGCTC TAGAATCACTTGTCAATTAATCCAGCTTCC) and cloned into pPur-LexA- Hnker using Agel and Xbal to create pPur-LexA-PCAF.

The LexA-Brm cassette is cut from pPur-LexA-Brm using ApaLI and EcoRI and blunted into the EcoRV site of PPLbs creating PPLBrm. P/CAF is cut from pPur-LexA-PCAF and cloned into PPLBrm using Agel and ApaLI/PacI creating PPLPCAF. The HAT domain of human p300 is pcr-amplified from plasmid pCMV6-p300 (Martinez-Balbas et al, 2000) using primers p300-a934F- Agel (GATCACCGGTCAGCCTGCAACTCCACTTTCC CAGCC) and p300- al652R-NheI (AGGCGCTAGCCTACATGGTGGACCACT GGGCTCTTCGG) and cloned into PPLBrm using Agel and Nhel/Xbal to create PPLp300 (FIG 1A). The HMTase domain of human Ashl is PCR amplified using primers HuAshl,aal787-For (GATCACCGGTACAAGCAGCTGTTCCCCCCATC ATATC) and HuAshl,aa2393-Rev (AGGCGCTAGCTCATAATGATGCTGAGT GAATATTATCAC) and cloned into Agel and Nhel digested PPLBrm to create PPLHuAshl. 5' STARs are cloned into the Sail site of the various PPL constructs. 3' STARs are cloned either into the Pad site (PPL, PPLBrm and PPLp300) or the Bsu36I site (PPLPCAF).

Transfection and culture of CHO cells The Chinese Hamster Ovary ceU line CHO-Kl (ATCC CCL-61) is cultured in HAMS-F12 medium + 10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicUlin, and 100 micrograms/ml streptomycin at 37 C/5% CO2. CeUs are transfected with the plasmids using SuperFect (QIAGEN) as described by the manufacturer. Briefly, ceUs are seeded to culture vessels and grown overnight to 70-90% confluence. SuperFect reagent is combined with plasmid DNA at a ratio of 6 microliters per microgram (e.g. for a 10 cm Petri dish, 20 micrograms DNA and 120 microhters SuperFect) and added to the cells. After overnight incubation the transfection mixture is replaced with fresh medium, and the transfected ceUs are incubated further. After overnight cultivation, cells are trypsinized and seeded into fresh culture vessels with fresh medium. After another overnight incubation zeocin is added to a concentration of 50 μg/ml and the cells are cultured further. After another three days the medium is replaced by fresh medium containing zeocin (100 μg/ml) and cultured further. When individual colonies become visible (approximately ten days after transfection) medium is removed and replaced with fresh medium without zeocin. Individual clones are isolated and transferred to 24-well plates in medium without zeocin. One day after isolation of the colonies zeocin is added to the medium. Expression of the GFP reporter gene is assessed approximately 3 weeks after transfection.

Example 2: Chromatin openers improve the level of transgene expression

One object of this invention is to improve both the predictabUity and the levels of transgene expression for heterologous protein production, thus increasing the yield of the heterologous protein and reducing the number of colonies that have to be analysed to obtain a high producer colony.

Materials and Methods The tested construct consist of a bicistronic gene with the GFP gene, an

IRES and the Zeocin resistance gene under control of the CMV promoter and a monocistronic gene encoding LexA-P/CAF under control of the SV40 promoter, but no STAR elements to flank the entire construct. The construct is transfected to CHO-Kl ceUs as in Example 1. Stable colonies are expanded before the GFP signal is determined on a XL-MCL Beckman Coulter flowcytometer. The mean of the GFP signal is taken as measure for the level of GFP expression and this is plotted in Figure 2. The results are compared to colonies that are transfected with a construct containing no LexA-P/CAF gene (Control) and a construct that is flanked with STAR 40 elements (STAR40- shielded) at both the 5' and 3' end, but that contains no LexA-P/CAF.

Results

FIG 2 shows that targeting LexA-P/CAF to LexA binding sites upstream of the CMV promoter results in a number of CHO colonies that express significantly higher levels of GFP protein, as compared to the "empty" control without LexA-P/CAF. The GFP signal in the colonies with the highest signals is comparable to the highest GFP signal that are obtained with a construct that has flanking STAR 40 elements, but no LexA-P/CAF. However, similar to the distribution of the GFP signals amongst the various colonies, most colonies do not express GFP or at a low level. This indicates that the predictabUity of the protein expression is not significantly altered as compared to the "empty" control construct. When compared to the GFP signals in colonies transfected with a STAR-shielded construct, these STAR elements convey a higher degree of predictabUity. The highest GFP expression level in STAR-shielded colonies is of the same order as the GFP expression level in LexA-P/CAF colonies. However, there are significantly more STAR-shielded colonies that show a high GFP expression level. It is therefore concluded that the LexA-P/CAF opener is able to convey higher expression levels to a transgene, but that they do not convey a higher predictabUity of transgene expression. Higher predictabUity is better achieved when STAR elements are added to a construct.

Example 3: The combination of chromatin openers and STAR elements improves predictability and yields of transgene expression

Openers are combined with STAR elements as described in Figure 1 and tested are the predictabUity and yield of transgene expression in stably transfected, individual colonies.

Materials and Methods

The tested construct consists of a bicistronic gene with the GFP gene, an IRES and the Zeocin resistance gene under control of the CMV promoter and a monocistronic gene encoding LexA-P/CAF under control of the SV40 promoter. The entire construct is flanked by STAR 40 (FIG. 1A). The construct is transfected to CHO-Kl ceUs as in Example 1. Stable colonies are expanded before the GFP signal is determined on a XL-MCL Beckman Coulter flowcytometer. The mean of the GFP signal is taken as measure for the level of GFP expression and this is plotted in Figure 3. The results are compared to colonies that are transfected with a construct containing no LexA-P/CAF gene and no STAR elements ("empty" control) and a construct that contains no LexA-P/CAF gene, but that is flanked with STAR 40 at both the 5' and 3' end.

Results

FIG 3 shows that the construct in which LexA- P/CAF is targeted to the CMV promoter and that is flanked by STAR elements conveys high GFP expression levels. The highest GFP expression level is more than three-fold higher than the highest levels in the "empty" control. Moreover, a high degree of predictabUity of GFP expression levels is found amongst various colonies. In contrast to colonies that express a construct with LexA-P/CAF alone (FIG. 2), more colonies that contain the construct with LexA-P/CAF and STAR40 elements have a high level of GFP expression. It is therefore concluded that the combination of STAR elements and an opener conveys both high protein expression levels and a high degree of predictabUity of expression.

Example 4: : Stability of transgene expression is improved by application of chromatin openers and STARs in expression systems. During cultivation of recombinant host ceUs, it is common practice to maintain antibiotic selection. This is intended to prevent transcriptional sUencing of the transgene, or loss of the transgene from the genome by processes such as recombination. However it is undesirable for production of proteins, for a number of reasons. First, the antibiotics that are used are quite expensive, and contribute significantly to the unit cost of the product. Second, for biopharmaceutical use, the protein must be demonstrably pure, with no traces of the antibiotic in the product. One advantage of STAR elements for heterologous protein production is that they confer stable expression on transgenes during prolonged cultivation, even in the absence of antibiotic selection; this property is demonstrated in this example and shown in Figure 5.

Materials and Methods

GFP expression levels in the colonies that are described in Example 2 and 3 are measured after periods of one week. After the initial three weeks after transfection when the first GFP measurements were performed, the colonies were cultured in medium without zeocin or other antibiotics. This continued for the remainder of the experiment. Results

FIG 4 shows the data on GFP expression of colonies that are stably transfected with the construct in which LexA-P/CAF is targeted to the CMV promoter and that is flanked by STAR elements. The colonies with the highest GFP expression levels in FIG 3 are chosen for analysis of stabUity of expression over time in the absence of selection pressure by antibiotics. The expression of the reporter GFP protein remains stable in the CHO ceUs in three time points. The first time point represents the start of the experiment when the selection pressure is removed. Measurements are performed after one, two and three weeks, which signifies approximately 10, 20 and 30 ceU cycles respectively. Colonies containing the STAR 40 and LexA-P/CAF are stable in the absence of antibiotics, but colonies containing only the LexA- P/CAF are not stable in the absence of antibiotics. This demonstrates that appHcation a combination of openers and STAR elements protect transgenes from sUencing during prolonged cultivation. It also demonstrates that this property is independent of antibiotic selection.

Example 5: The p300HAT chromatin opener has no effect on transient gene expression driven by the CMV and UB6 promoters, but does have an effect on a minimal promoter.

The nature of the effects of openers on gene expression is investigated. One possible way of action is that the openers act in a transient manner on promoters and that this effect is subsequently transmitted to stable clones. Hence we tested the effects of several openers on transient expression levels. Promoters used are the strong CMV and UB6 promoters, as weU as the minimal SV40 promoter.

Materials and Methods Plasmids The reporter constructs consist of the DsRED gene under the control of either the CMV, UB6 or the minimal SV40 promoter. Upstream of these promoters are LexA binding sites. LexA binding sites (LBS) are amplified from plasmid pREP4-HSF-Luc+ (van der Vlag et al, 2000) using primers LBS-for-Sall (AGGCGTCGACGTTTCG ACTCCCAAGCTTTG) and LBS-rev-AscI

(GATCGGCGCGCCGGTACC ATAGCGGCCGCGAC) and cloned upstream of the promoters in PP using Sail and Ascl. Another construct contains a gene encoding a portion of the p300 gene, encoding the histone acetyltransferase domain (HAT). The HAT domain of human p300 is pcr-amplified from plasmid pCMV6-p300 (Martinez-Balbas et al, 2000) using primers p300-a934F-AgeI (GATCACCGGTCAGCCTGCAACTCCACTTTCCCAGCC) and _P300-al652R- Nhel (AGGCGCTAGCCTACATGGTGGACCACTGGGCTCTTCGG) and cloned using Agel and Nhel/Xbal to create PPLp300HAT. The p300HAT domain is cloned in frame with the LexA protein and the entire cassette is placed under control of the SV40 promoter.

Transfection and culture of CHO cells

The Chinese Hamster Ovary cell line CHO-Kl (ATCC CCL-61) is cultured in

HAMS-F12 medium + 10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicilHn, and 100 micrograms/ml streptomycin at 37 C/5% CO2. Cells are transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. The DsRED reporter gene with the different promoters is transfected either alone or in combination with the SV40-p300HAT construct. Transfected cells are seeded in culture vessels and grown overnight to 70-90% confluence. Lipofectamine reagent is combined with plasmid DNA at a ratio of 7.5 microhters per 3 microgram and added after a 30 minutes incubation at 25°C to the ceUs. After 6 hour incubation the transfection mixture is replaced with fresh medium, and the transfected ceUs are incubated further. 24 hours after transfection the DsRED signal is determined on a XL-MCL Beckman Coulter flowcytometer. The mean of the DsRED signal is taken as measure for the level of DsRED expression and this is plotted in Figure 5.

Results FIG 5 shows that targeting LexA-P300 HAT to LexA binding sites upstream of the CMV or the UB6 promoter does not result in an increase of DsRED expression. However, when the Lex-P300HAT is expressed with the plasmid containing the DsRED gene driven by the SV40 minimal promoter, an increase of 400% is seen. Thus, the Lex-P300HAT does not enhance transient expression levels of CMV and UB6 driven gene expression, but only of a minimal promoter, in this case the SV40 minimal promoter.

Example 6: The p300HAT opener improves the level of CMV-driven expression in stably transfected clones, but only for a limited period. During cultivation of recombinant host cells, it is common practice to maintain antibiotic selection. This is intended to prevent transcriptional sUencing of the transgene, or loss of the transgene from the genome by processes such as recombination. However it is undesirable for production of proteins, for a number of reasons. First, the antibiotics that are used are quite expensive, and contribute significantly to the unit cost of the product. Second, for biopharmaceutical use, the protein must be demonstrably pure, with no traces of the antibiotic in the product. In this example we test whether the P300HAT Opener is able to induce stabUity of gene expression over an extended period of time.

Materials and Methods

Plasmids

Two plasmids are compared in this experiment: the CMV-d2EGFP-ires-Zeo

(CMV Control) vector and the CMV-d2EGFP-ires-Zeo- LexA-P300HAT (CMV- p300HAT) (FIG6). The open reading frame of the zeocin-resistance gene is inserted into BamΗI/Notl sites downstream of the pIRES as foUows: the zeocin-resistance ORF is amplified by PCR with primers GATCGGATCCTTCGAAATGGC CAAGTTGACCAGTGC and AGGCGCGGCCGCAATTCTCAGTCCT GCTCCTC from plasmid pEM7/zeo, digested with _Bα HI and Notl, and Hgated with £αm.HI/iVotI-digested pIRES- hnk to yield pIRES-Hnk-zeo. The d2EGFP reporter ORF was introduced into pIRES-Hnk-zeo by amplification of (Clontech 6010-1) with primers GATCGAATTCTCGCGAATGGTGAGCAAGCAG ATCCTGAAG and AGGCGAATTCACCGGTGTTTAAACTTACACCCACTC GTGCAGGCTGCCCAGG, and insertion of the EcoRI-digested d2EGFP cassette into the .EcoRI site in the pIRES-Hnk-zeo plasmid. This created the CMV Control (CMV-d2EGFP-IRES-Zeo).

The effect of the LexA-P300HAT on gene expression is determined with a plasmid that differs significantly from the plasmids in FIG1A. In FIG1A the SV40-Lex-Opener unit is placed downstream from the other expression unit that encompasses the CMV-driven GFP reporter gene. Transcription of both units is then in the same direction. In the novel plasmid (FIG 6), the transcription of the CMV-driven d2EGFP reporter gene is directed away from the transcription of the SV40-driven LexA-P300HAT opener. In this configuration the CMV and SV40 promoters are in close proximity. Between these two promoters LexA binding sites are cloned. Hence the LexA-P300HAT will influence the expression status of both the CMV-driven reporter gene and the SV40-driven Opener. LexA binding sites (LBS) are amphfied from plasmid pREP4-HSF-Luc+ (van der Vlag et al, 2000) using primers AGGCGTCGACGTTTCG ACTCCCAAGCTTTG and

GATCGGCGCGCCGGTACC ATAGCGGCCGCGAC and cloned between the CMV and SV40 promoters in PP using Sail and Ascl.

LexA is amplified from plasmid pEG202 (Bennetzen and HaU, 1982) using primers GATCAAGCTTATGAAGACGTTAACGGCCAGGC and AGGCACCGGTCAGCCAGTCGCCGTTGCGAATAACC and cloned downstream of the SV40 promoter in plasmid pPur using Hindlll and Agel creating pPur-LexA. OHgo's GATCTCCCCTGAGGAAGTGCACAACCTGA GGCC and GATCTGGCCTCAGGTTGTGCACT TCCTCAGGGG are Hgated into the BamHI site of pPur-LexA to create pPur-LexA-Hnker. The HAT domain of human p300 (aa934-1652) is pcr-amplified from plasmid pCMVB- p300 (Martinez-Balbas et al, 2000) using primers GATCACCGGTCAGCCT GCAACTCCACTTTCCCAGCC and AGGCGCTAGCCTACATGGTGG ACCACTGGGCTCTTCGG and cloned into pPur-LexA-Hnker using Agel and Nhel/Xbal creating pPur-LexA-P300-HAT. The entire SV40-LexA-P300-HAT transcription unit is cloned downstream of the LexA binding sites, to create — CMV-d2EGFP-ires-Zeo-LexA-P300HAT (CMV-p300HAT) (FIG6).

Transfection and culture of CHO cells

The Chinese Hamster Ovary ceU Hne CHO-Kl (ATCC CCL-61) is cultured in HAMS-F12 medium + 10% Fetal Caff Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37 C/5% CO2. Cells are transfected with the plasmids using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Briefly, cells are seeded in culture vessels and grown overnight to 70-90% confluence. Lipofectamine reagent is combined with plasmid DNA at a ratio of 7.5 microhters per 3 microgram and added after a 30 minutes incubation at 25°C to the ceUs. After 6 hours incubation the transfection mixture is replaced with fresh medium, and the transfected cells are incubated further. After overnight cultivation, ceUs are trypsinized and seeded in serial dUutions into fresh petri dishes with fresh medium with zeocin added to a concentration of 100 μg/ml and the ceUs are cultured further. When individual colonies become visible (approximately ten to fourteen days after transfection). Individual clones are isolated and transferred to 24-weU plates in medium containing zeocin. Expression of the d2EGFP reporter gene is assessed approximately four weeks after transfection. After these first measurements cells are subsequently cultured in medium without selection agent (Zeocin). Expression of the d2EGFP reporter gene is assessed at different times, up to 120 days post-transfection.

Results FIG 6 shows that targeting LexA-P300HAT to LexA binding sites upstream of the CMV promoter results in a number of CHO colonies that express shghtly higher levels of d2EGFP protein, as compared to the "empty" control without LexA-P300HAT. The average of the d2EGFP signal in the 11 colonies transfected with the CMV Control plasmid is 109, when measured 30 days after transfection. In comparison, the average of the d2EGFP signal in the 15 colonies transfected with the CMV-p300HAT plasmid is 158, when measured 30 days after transfection. However, when foUowed for an extended period of time, expression levels of both plasmids dropped. The average of the d2EGFP signal in the 11 colonies transfected with the CMV Control plasmid is 28, when measured 120 days after transfection. The average of the d2EGFP signal in the 15 colonies transfected with the CMV-p300HAT plasmid is 70, when measured 120 days after transfection. Hence the expression level of the plasmid without the LexA-P300HAT Opener dropped a factor four, whereas the expression level of the plasmid with the LexA-P300HAT Opener dropped a factor of two. We conclude that the LexA-p300HAT conveys more stabUity of gene expression levels in comparison with the plasmid without Opener, but this only to a Hmited degree. With the CMV promoter the higher expression levels induced by the LexA-P300HAT Opener is therefore limited in time, at least when the cells are cultured in the absence of antibiotic selection pressure.

Example 7: STAR and TRAP elements improve stability over time of the p300 HAT-mediated increased gene expression levels

In this example we test whether the combination of STAR, TRAP elements and the LexA-P300HAT is able to promote long-term stabihty of gene expression. Materials and Methods Plasmids

Two plasmids are compared in this experiment: the UB6-d2EGFP-ires-Zeo (UB6 Control) vector and the UB6-d2EGFP-ires-Zeo-LexAP300HAT-STAR7- TRAP (UB6-p300HAT-STAR7) (FIG7). The configuration of the Opener element in relation to the transcription unit of the reporter gene is similar as in FIG 6. However, the CMV promoter is replaced by the UB6 promoter (PCR- amplified from pUB6V5HisA using primers GATCGGTACC GGCGCGCCTCCGCGCCGGGTTTTG and AGGCGAGCTCGGTACCAAGC TTCGTCTAAC and cloned into PPLp300HAT using Ascl and Sad creating UB6-p300HAT). Also, 5' STAR7 is cloned into the Sail site and 3' STAR7 is cloned into the Pad site of the UB6-p300HAT construct to create UB6- p300HAT-STAR7 To the STAR7 sequence the SPA-pause TRAP sequence is added. (FIG 7).

Transfection and culture of CHO cells

Transfection and culturing are as in Example 6. Expression of the d2EGFP reporter gene is assessed approximately three weeks after transfection. After these first measurements ceUs are subsequently cultured in medium without selection agent (Zeocin). Expression of the d2EGFP reporter gene is assessed at different times, for the last time 95 days after transfection.

Results FIG 7 shows that targeting LexA-P300HAT to LexA binding sites upstream of the UB6 promoter results in a number of CHO colonies that express significantly higher levels of d2EGFP protein, as compared to the "empty" UB6 Control without LexA-P300HAT. The average of the d2EGFP signal in the 12 colonies transfected with the UB6 Control plasmid is 70, when measured 25 days after transfection. In comparison, the average of the d2EGFP signal in the 12 colonies transfected with the UB6-p300HAT-STAR7 plasmid is 157, when measured 25 days after transfection. Hence the combined application of STAR7 and the LexA-P300HAT Opener has a positive effect on the expression level of the reporter protein in stably transfected clones. When foUowed for an extended period of time, only the expression levels of the UB6 control plasmid dropped. The average of the d2EGFP signal in the 12 colonies transfected with the UB6 Control plasmid is 31, when measured 95 days after transfection. The average of the d2EGFP signal in the 12 colonies transfected with the UB6-p300HAT-STAR7 plasmid is 207, when measured 95 days after transfection. Hence the expression level of the plasmid without the LexA-P300HAT Opener dropped significantly, whereas the expression level of the plasmid with the LexA-P300HAT Opener has actuaUy increased ~30%. We conclude that the combined action of STAR7, the Spa/pause TRAP sequence and the LexA-p300HAT Opener conveys a high degree of stability of gene expression levels in comparison with the plasmid without STAR, TRAP sequences and an Opener. At least in context of the UB6 promoter, the higher expression levels induced by STAR7/TRAP and the LexA-P300HAT Opener remains therefore stable over time. This demonstrates that apphcation of a combination of Openers, STAR and TRAP elements protect transgenes from sUencing during prolonged cultivation. It also demonstrates that this property is independent of antibiotic selection.

Example 8: The combination of the p300 HAT opener and a STAR/TRAP element improves copy number dependency of gene expression

Transgene expression units for heterologous protein expression are generally integrated into the genome of the host cell to ensure stable retention during cell division. Integration can result in one or multiple copies of the expression unit being inserted into the genome; multiple copies may or may not be present as tandem arrays. This raises the question whether the transgene is expressed in copy-number dependent or independent fashion. Particularly clones containing a higher copy-number tend to express unstable over time. In this example we determine the relationship between transgene expression levels and copy number.

Material and methods

CHO ceUs were transfected with UB6-d2EGFP-ires-Zeo (UB6 Control) vector and the UB6-d2EGFP-ires-Zeo-LexA-P300HAT-STAR7-TRAP (UB6-p300HAT- STAR7). Individual clones were selected and were cultivated for 95 days, as in Example 7. CeUs were harvested, d2EGFP expression was measured and the remaining ceUs were lysed and the genomic DNA purified using the DNeasy Tissue Kit (QIAGEN 69504) as described by the manufacturer. The copy number of the d2EGFP gene was determined by foUowing a competitive PCR protocol (Fu et al., 1999). The resulting autoradiogram exposed to a phosphorimager screen (Personal F/X, BioRad) and was analysed by densitometry to determine the relative strength of the d2EGFP DNA bands. The blot was re-hybridized with a probe for actin and the ratio between the d2EGFP and actin signal was taken as the relative copy number.

Results

We determined the copy-number of the integrated vectors in the colonies shown in FIG. 8. No correlation between the copy- number of integrated plasmids and the expression level of d2EGFP in the UB6 Control vector, in which no STAR element was present. This is indicated by the low correlation coefficient (R²) of 0.24. In contrast, there was a good correlation between expression and copy-number of d2EGFP in the UB6-p300HAT-STAR7 vector. This is indicated by the high correlation coefficient (R²) of 0.82. Significantly, not only was the d2EGFP expression copy-number dependent, more d2EGFP protein was produced per copy. An estimate is that there is a factor 3 increased d2EGFP expression level per copy in the UB6-p300HAT-STAR7 vector. This suggests that the combination of STAR7, the Spa/pause TRAP sequence and the LexA-P300HAT Opener confers copy number-dependence on the transgene expression units, making transgene expression independent of other transgene copies in tandem arrays, and independent of gene-sUencing influences at the site of integration. Furhermore, expression per copy increases when the transgene is protected by the combination of STAR7/TRAP and the LexA-P300HAT Opener.

DESCRIPTION OF FIGURES

FIG 1. Schematic diagram of the invention.

FIG 1A shows two expression units on one plasmid. Expression unit one comprises a bicistronic gene containing (from 5' to 3') a transgene (encoding for example one subunit of a multimeric protein; Genel), an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the CMV promoter. Upstream of the CMV promoter are four LexA binding sites (LexA- BS). The expression unit has the SV40 transcriptional terminator at its 3' end (t). Next is a monocistronic gene encoding a fusion protein between the LexA protein and either (i) a histone acetyltransferase (HAT) or a functional part of a HAT that is still able to transfer acetyl groups to histone taUs (LexA-HAT), (H) a histone methyltransferase (HMTase) or a functional part (SET domain) of a HMTase that is stUl able to transfer methyl groups to at least lysine K4 of the histone H3 taU or (Hi) the trithorax group protein Brahma. These genes are under control of the SV40 promoter. The expression unit has the SV40 transcriptional terminator at its 3' end (t). The entire cassette with the two expression units is flanked by STAR elements.

FIG IB is similar to FIG 1A, but there are now three expression units on one plasmid. Expression unit one comprises a bicistronic gene containing a transgene Genel, an IRES, and a selectable marker zeo under control of the CMV promoter. The transcription orientation of this first expression unit is directed upstream. Expression unit two comprises a bicistronic gene containing a transgene Gene2, an IRES, and a selectable marker puro (puromycin resistance gene) under control of the CMV promoter. The transcription orientation of this first expression unit is directed downstream. Between the two CMV promoter of the two expression units are four LexA binding sites (LexA-BS). The monocistronic gene encodes the same LexA fusion proteins as in FIG 1A. The entire consteUation of three expression units is flanked by STAR elements. FIG 2. Chromatin openers improve CMV driven GFP expression in CHO cells

The constructs that contain the gene encoding LexA-P/CAF are transfected to CHO-Kl ceUs. Stable colonies (14 of each construct) are expanded and the GFP signal is determined on a XL-MCL Beckman Coulter flowcytometer. For each independent colony the mean of the GFP signal is plotted. This is taken as measure for the level of GFP expression. The results are compared to colonies that are transfected with a construct containing no LexA-P/CAF gene (Control) and a construct that is flanked with STAR 40 elements (STAR40-shielded) at both the 5' and 3' end.

FIG 3. The combination of chromatin openers and STARs enhances CMV promoter driven GFP expression in CHO cells The construct that is flanked by STAR 40 and that contains the gene encoding LexA-P/CAF (see FIG 1) is transfected to CHO-Kl ceUs. Stable colonies (14 of each construct) are expanded, the GFP signal is determined and the mean of the GFP signal is plotted as in FIG 2. The results are compared to colonies that are transfected with a construct containing no LexA-P/CAF or STAR 40 elements (Control) and a construct that is flanked with STAR 40 elements (STAR40) at both the 5' and 3' end.

FIG 4. The combination of chromatin openers and STARs enhances the stability of CMV promoter driven GFP expression in CHO cells Stably transfected colonies that contain either the LexA-P/CAF opener construct or the GFP construct that is flanked by STAR 40 as weU contains the gene encoding LexA- P/CAF (see FIG 1) are expanded. Of both categories four colonies are chosen with the highest GFP levels (see FIG 3). These colonies are further cultured without the antibiotic (zeocin) and the GFP signal is determined with intervals of one week, which represent approximately 10 ceU cycles. The mean of the GFP signal is plotted as in FIG 3. The first bar of each colony represents the GFP signal at the moment that the antibiotic selection pressure is removed. The adjacent three bars represent the GFP signal that is measured after one, two and three weeks.

FIG 5. LexA-P300HAT does not enhance transient expression of the CMV and UB6 promoter, but only of the minimal SV40 promoter. FIG 5 shows two different classes of plasmids. Class one comprises the DsRED reporter gene under control of the CMV, UB6 or minimal SV40 promoter. Upstream of these promoters are four LexA binding sites (LexA-BS). The second class of plasmid is a gene encoding a fusion protein between the LexA protein and the functional P300 histone acetyltransferase (HAT) domain. The different reporter gene constructs are transfected alone or together with the plasmid encoding the LexA-P300HAT Opener protein. 24 hours afters transfection the DsRED signals are measured. The signal measured with only the reporter gene construct is set arbitrarUy at 100. The signal of such a reporter gene alone is compared with the signal of that respective reporter gene construct in combination with the LexA-P300HAT Opener construct.

FIG 6. The p300HAT opener improves the level of CMV-driven expression in stably transfected clones, but only for a limited period.

Two different constructs are transfected to CHO-Kl ceUs. Construct one comprises a bicistronic gene containing (from 5' to 3') the d2EGFP reporter gene, an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the CMV promoter (CMV Control).

Construct two comprises a first expression unit comprising a bicistronic gene containing (from 5' to 3') the d2EGFP reporter gene, an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the CMV promoter. The cassette has the SV40 transcriptional terminator at its 3' end (t). Upstream of the CMV promoter are four LexA binding sites (LexA-BS). Downstream of the LexA binding sites is a second expression unit, a monocistronic gene encoding a fusion protein between the LexA protein and the functional P300 histone acetyltransferase (HAT) domain. The expression unit has the SV40 transcriptional terminator at its 3' end (t). Transcription of both expression units is directed opposite. Hence the LexA binding sites are placed between and wUl act upon both expression units (CMV-p300HAT).

An indicated number of stable colonies are expanded and after different, indicated time periods, the d2EGFP signal is determined on a XL-MCL Beckman Coulter flowcytometer. For each independent colony the mean of the d2EGFP signal is plotted. This is taken as measure for the level of d2EGFP expression. The results are compared to colonies that are transfected with a construct containing no LexA-P300HAT gene (CMV- Control).

FIG 7. The increased gene expression levels due to the combined action of STAR7/TRAP and the p300 HAT opener is highly stable over time.

Two different constructs are transfected to CHO-Kl ceUs. Construct one comprises a bicistronic gene containing (from 5' to 3') the d2EGFP reporter gene, an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the UB6 promoter (UB6 Control).

Construct two comprises a first expression unit comprising a bicistronic gene containing (from 5' to 3') the d2EGFP reporter gene, an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the UB6 promoter. The cassette has the SV40 transcriptional terminator at its 3' end (t). Upstream of the CMV promoter are four LexA binding sites (LexA-BS). Downstream of the LexA binding sites is a second expression unit, a monocistronic gene encoding a fusion protein between the LexA protein and the functional P300 histone acetyltransferase (HAT) domain. The expression unit has the SV40 transcriptional terminator at its 3' end (t). Transcription of both expression units is directed opposite. Hence the LexA binding sites are placed between and wUl act upon both expression units. The entire cassette is flanked with STAR 7 elements (STAR7) at both the 5' and 3' end (UB6- p300HAT-STAR7). To the STAR7 sequence a SPA-pause TRAP sequence is added. An indicated number of stable colonies are expanded and after different, indicated time periods, the d2EGFP signal is determined on a XL-MCL Beckman Coulter flowcytometer. For each independent colony the mean of the d2EGFP signal is plotted. This is taken as measure for the level of d2EGFP expression. The results are compared to colonies that are transfected with a construct containing no LexA-P300HAT gene (UB6-Control).

FIG 8. The combination of the p300 HAT opener and a STAR/TRAP element improves copy number dependency of gene expression d2EGFP expression units in UB6 Control (A) and UB6-p300HAT-STAR7 (B), integrated into CHO genomic DNA are analysed for d2EGFP expression and the presence of the number of d2EGFP copies. Radioactive d2EGFP DNA probe was used to detect the amount of transgene DNA in the genome of each clone, which was then quantified with a phosphorimager. The expression of the clones are plotted against the relative copy number. The correlation coefficient is indicated in each case.

Instead of LexA-Opener fusion proteins that targeted to LexA binding sites, also GAL4-Opener fusion proteins can be used. These GAL4-Opener fusion proteins are targeted to GAL4 binding sites, which are placed upstream of a promoter. UnHke the bacterial LexA protein, GAL4 is a yeast protein. Like LexA protein, GAL4 is a transcription factor that has a DNA binding domain and a trans-acting domain, the last domain being responsible for activation of gene expression. To create a GAL4-Opener fusion protein, the part of the GAL4 gene, encoding amino acids 1 to 147 (Lillie and Green, 1989), is cloned in frame with the respective Opener protein or functional part of the Opener protein. In the current invention expression of the GAL4-Opener fusion gene is driven by the SV40 promoter. The GAL4-Opener fusion protein is targeted to GAL4 binding sites, caUed GAL4 operators. Commonly four GAL4 operators are placed immediately upstream of a promoter. One GAL4 operator is the foUowing sequence: CGGAGTACTGTCCTCCG.

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SEQ ID: 1 >STAR4

GATCTGAGTCATGTTTTAAGGGGAGGATTCTTTTGGCTGCTGAGTTGAGATTAGGTTGAGGGTAGTG AAGGTAAAGGCAGTGAGACCACGTAGGGGTCATTGCAGTAATCCAGGCTGGAGATGATGGTGGTTC AGTTGGAATAGCAGTGCATGTGCTGTAACAACCTCAGCTGGGAAGCAGTATATGTGGCGTTATGACC TCAGCTGGAACAGCAATGCATGTGGTGGTGTAATGACCCCAGCTGGGTAGGGTGCATGTGATGGAA CAACCTCAGCTGGGTAGCAGTGTACTTGATAAAATGTTGGCATACTCTAGATTTGTTATGAGGGTAG TGCCATTAAATTTCTCCACAAATTGGTTGTCACGTATGAGTGAAAAGAGGAAGTGATGGAAGACTTC AGTGCTTTTGGCCTGAATAAATAGAAGACGTCATTTCCAGTTAATGGAGACAGGGAAGACTAAAGGT AGGGTGGGATTCAGTAGAGCAGGTGTTCAGTTTTGAATATGATGAACTCTGAGAGAGGAAAAACTTT TTCTACCTCTTAGTTTTTGTGACTGGACTTAAGAATTAAAGTGACATAAGACAGAGTAACAAGACAAA AATATGCGAGGTTATTTAATATTTTTACTTGCAGAGGGGAATCTTCAAAAGAAAAATGAAGACCCAAA GAAGCCATTAGGGTCAAAAGCTCATATGCCTTTTTAAGTAGAAAATGATAAATTTTAACAATGTGAGA AGACAAAGGTGTTTGAGCTGAGGGCAATAAATTGTGGGACAGTGATTAAGAAATATATGGGGGAAAT GAAATGATAAGTTATTTTAGTAGATTTATTCTTCATATCTATTTTGGCTTCAACTTCCAGTCTCTAGTG ATAAGAATGTTCTTCTCTTCCTGGTACAGAGAGAGCACCTTTCTCATGGGAAATTTTATGACCTTGCT GTAAGTAGAAAGGGGAAGATCTCCTGTTTCCCAGCATCAGGATGCAAACATTTCCCTCCATTCCAGT TCTCAACCCCATGGCTGGGCCTCATGGCATTCCAGCATCGCTATGAGTGCACCTTTCCTGCAGGCTG CCTCGGGTAGCTGGTGCACTGCTAGGTCAGTCTATGTGACCAGGAGCTGGGCCTCTGGGCAATGCC AGTTGGCAGCCCCCATCCCTCCACTGCTGGGGGCCTCCTATCCAGAAGGGCTTGGTGTGCAGAACGA TGGTGCACCATCATCATTCCCCACTTGCCATCTTTCAGGGGACAGCCAGCTGCTTTGGGCGCGGCAA AAAACACCCAACTCACTCCTCTTCAGGGGCCTCTGGTCTGATGCCACCACAGGACATCCTTGAGTGC TGGGCAGTCTGAGGACAGGGAAGGAGTGATGACCACAAAACAGGAATGGCAGCAGCAGTGACAGGA GGAAGTCAAAGGCTTGTGTGTCCTGGCCCTGCTGAGGGCTGGCGAGGGCCCTGGGATGGCGCTCAG TGCCTGGTCGGCTGCAAGAGGCCAGCCCTCTGCCCATGAGGGGAGCTGGCAGTGACCAAGCTGCAC TGCCCTGGTGGTGCATTTCCTGCCCCACTCTTTCCTTCTAAGATC

SEQ ID:2 >STAR6

GATCTGACCCACCACAGACATCCCCTCTGGCCTCCTGAGTGGTTTCTTCAGCACAGCTTCCAGAGCC AAATTAAACGTTCACTCTATGTCTATAGACAAAAAGGGTTTTGACTAAACTCTGTGTTTTAGAGAGGG AGTTAAATGCTGTTAACTTTTTAGGGGTGGGCGAGAGGAATGACAAATAACAACTTGTCTGAATGTT TTACATTTCTCCCCACTGCCTCAAGAAGGTTCACAACGAGGTCATCCATGATAAGGAGTAAGACCTC CCAGCCGGACTGTCCCTCGGCCCCCAGAGGACACTCCACAGAGATATGCTAACTGGACTTGGAGACT GGCTCACACTCCAGAGAAAAGCATGGAGCACGAGCGCACAGAGCAGGGCCAAGGTCCCAGGGACAG AATGTCTAGGAGGGAGATTGGGGTGAGGGTAATCTGATGCAATTACTGTGGCAGCTCAACATTCAAG GGAGGGGGAAGAAAGAAACAGTCCCTGTCAAGTAAGTTGTGCAGCAGAGATGGTAAGCTCCAAAAT TTGAAACTTTGGCTGCTGGAAAGTTTTAGGGGGCAGAGATAAGAAGACATAAGAGACTTTGAGGGTT TACTACACACTAGACGCTCTATGCATTTATTTATTTATTATCTCTTATTTATTACTTTGTATAACTCTT ATAATAATCTTATGAAAACGGAAACCCTCATATACCCATTTTACAGATGAGAAAAGTGACAATTTTGA GAGCATAGCTAAGAATAGCTAGTAAGTAAAGGAGCTGGGACCTAAACCAAACCCTATCTCACCAGAG TACACACTCTTTTTTTATTCCAGTGTAATTTTTTTTAATTTTTATTTTACTTTAAGTTCTGGGATACAT GTGCAGAAGGTATGGTTTGTTACATAGGTATATGTGTGCCATAGTGGATTGCTGCACCTATCAACCC GTCATCTAGGTTTAAGCCCCACATGCATTAGCTATTTGTCCTGATGCTCTCCCTCCCCTCCCCACACC AGACAGGCCTTGGTGTGTGATGTTCCCCTCCCTGTGTCCATGTGTTCTCACTGTTCAGCTCCCACTTA TGAGTGAGAACATGTGGTATTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAGGATGATGGCTTCCAGC TTCATCCATGTCCCTGCAAAGGACACGATC

SEQ ID:3 >STAR7

GATCACCCGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGTAAAACCTCGTCTCTACTAAAAA AATACGAAAAATTAGCTGGTTGTGGTGGTGCGTGCTTGTAATCCCAGCTACTCGGGAGGCTGAGGCA GGAGAATCACTTGAATCTGGGAGGCAGAGGTTGCAGTGAGCTGAGATAGTGCCATTGCACTCCAGC CTGGGCAACAGACGGAGACTCTGTCTCCAAAAAAAAAAAAAAAAATCTTAGAGGACAAGAATGGCTC TCTCAAACTTTTGAAGAAAGAATAAATAAATTATGCAGTTCTAGAAGAAGTAATGGGGATATAGGTG CAGCTCATGATGAGGAAGACTTAGCTTAACTTTCATAATGCATCTGTCTGGCCTAAGACGTGGTGAG CTTTTTATGTCTGAAAACATTCCAATATAGAATGATAATAATAATCACTTCTGACCCCCCTTTTTTTTC CTCTCCCTAGACTGTGAAGCAGAAACCCCATATTTTTCTTAGGGAAGTGGCTACGCACTTTGTATTTA TATTAACAACTACCTTATCAGGAAATTCATATTGTTGCCCTTTTATGGATGGGGAAACTGGACAAGTG ACAGAGCAAAATCCAAACACAGCTGGGGATTTCCCTCTTTTAGATGATGATTTTAAAAGAATGCTGC CAGAGAGATTCTTGCAGTGTTGGAGGACATATATGACCTTTAAGATATTTTCCAGCTCAGAGATGCT ATGAATGTATCCTGAGTGCATGGATGGACCTCAGTTTTGCAGATTCTGTAGCTTATACAATTTGGTGG TTTTCTTTAGAAGAAAATAACACATTTATAAATATTAAAATAGGCCCAAGACCTTACAAGGGCATTCA TACAAATGAGAGGCTCTGAAGTTTGAGTTTGTTCACTTTCTAGTTAATTATCTCCTGCCTGTTTGTCA TAAATGCGTTTAGTAGGGAGCTGCTAATGACAGGTTCCTCCAACAGAGTGTGGAAGAAGGAGATGAC GGCTGGCTTCCCCTCTGGGACAGCCTCAGAGCTAGTGGGGAAACTATGTTAGCAGAGTGATGCAGT GACCAAGAAAATAGCACTAGGAGAAAGCTGGTCCATGAGCAGCTGGTGAGAAAAGGGGTGGTAATC ATGTATGCCCTTTCCTGTTTTATTTTTTATTGGGTTTCCTTTTGCCTCTCAATTCCTTCTGACAATACA AAATGTTGGTTGGAACATGGAGCACCTGGAAGTCTGGTTCATTTTCTCTCAGTCTCTTGATGTTCTCT CGGGTTCACTGCCTATTGTTCTCAGTTCTACACTTGAGCAATCTCCTCAATAGCTAAAGCTTCCACAA TGCAGATTTTGTGATGACAAATTCAGCATCACCCAGCAGAACTTAGGTTTTTTTCTGTCCTCCGTTTC CTGACCTTTTTCTTCTGAGTGCTTTATGTCACGTCGTGAACCATCCTTTCCTTAGTCATCTACCTAGC AGTCCTGATTCTTTTGACTTGTCTCCCTACACCACAATAAATCACTAATTACTATGGATTCAATCCCT AAAATTTGCACAAACTTGCAAATAGATTACGGGTTGAAACTTAGAGATTTCAAACTTGAGAAAAAAGT TTAAATCAAGAAAAATGACCTTTACCTTGAGAGTAGAGGCAATGTCATTTCCAGGAATAATTATAATA ATATTGTGTTTAATATTTGTATGTAACATTTGAATACCTTCAATGTTCTTATTTGTGTTATTTTAATCT CTTGATGTTACTAACTCATTTGGTAGGGAAGAAAACATGCTAAAATAGGCATGAGTGTCTTATTAAAT GTGACAAGTGAATAGATGGCAGAAGGTGGATTCATATTCAGTTTTCCATGACCCTGGAAATCATGCG GAGATGATTTCTGCTTGCAAATAAAACTAACCCAATGAGGGGAACAGCTGTTCTTAGGTGAAAACAA AACAAACACGCCAAAAACCTTTATTCTCTTTATTATGAATCAAATTTTTCCTCTCAGATAATTGTTTTA TTTATTTATTTTTATTATTATTGTTATTATGTCCAGTCTCACTCTGTCGCCTAAGCTGGCATGATC

SEQ ID:4

>STAR12

ATCCTGCTTCTGGGAAGAGAGTGGCCTCCCTTGTGCAGGTGACTTTGGCAGGACCAGCAGAAACCCA

GGTTTCCTGTCAGGAGGAAGTGCTCAGCTTATCTCTGTGAAGGGTCGTGATAAGGCACGAGGAGGC AGGGGCTTGCCAGGATGTTGCCTTTCTGTGCCATATGGGACATCTCAGCTTACGTTGTTAAGAAATA TTTGGCAAGAAGATGCACACAGAATTTCTGTAACGAATAGGATGGAGTTTTAAGGGTTACTACGAAA AAAAGAAAACTACTGGAGAAGAGGGAAGCCAAACACCACCAAGTTTGAAATCGATTTTATTGGACGA ATGTCTCACTTTAAATTTAAATGGAGTCCAACTTCCTTTTCTCACCCAGACGTCGAGAAGGTGGCATT CAAAATGTTTACACTTGTTTCATCTGCCTTTTTGCTAAGTCCTGGTCCCCTACCTCCTTTCCCTCACTT CACATTTGTCGTTTCATCGCACACATATGCTCATCTTTATATTTACATATATATAATTTTTATATATGG CTTGTGAAATATGCCAGACGAGGGATGAAATAGTCCTGAAAACAGCTGGAAAATTATGCAACAGTGG GGAGATTGGGCACATGTACATTCTGTACTGCAAAGTTGCACAACAGACCAAGTTTGTTATAAGTGAG GCTGGGTGGTTTTTATTTTTTCTCTAGGACAACAGCTTGCCTGGTGGAGTAGGCCTCCTGCAGAAGG CATTTTCTTAGGAGCCTCAACTTCCCCAAGAAGAGGAGAGGGCGAGACTGGAGTTGTGCTGGCAGCA CAGAGACAAGGGGGCACGGCAGGACTGCAGCCTGCAGAGGGGCTGGAGAAGCGGAGGCTGGCACC CAGTGGCCAGCGAGGCCCAGGTCCAAGTCCAGCGAGGTCGAGGTCTAGAGTACAGCAAGGCCAAGG TCCAAGGTCAGTGAGTCTAAGGTCCATGGTCAGTGAGGCTGAGACCCAGGGTCCAATGAGGCCAAG GTCCAGAGTCCAGTAAGGCCGAGATC

SEQ ID:5 >STAR18

CTAAAGGCATTTTATATAGAGCTGTGGTTTTTGTGGTTTACCTGTGGCCGTGGCCAGAGGTTCCTGG GAGGCTAACAGGTGTTTTTTGAGGGTTGGGGCTTGGGTGGGGGTGGGGTGAATTCTCTGTTTCTAGG ATGTGCTTGGTGTTTGAATCTAGGCTTTAGTGACTGATGCTGGTTAATTTCTAGGGTTGATGGTTTAT TGGGCCTTGTGTTGTATGAGATGGAATTTTAAATATTTTTAAATGTTTCTCTAGTTCTTAGAGAAATT TTTAAGCAACTCAAGATAGGCTCTTCCCGCATATGATAATCCGTCAGGTGAATTTGGATTCTTTTATA TCACAAAATGAATCCATGTTTTGGGAGGTAATGGTATCAGAATATATGGTGCAGGTCTTGGTAAAAA CCCAATAGATCTTTGAGAAATACAAGACATCTCTGTGTTGAAACATCGTGTGTTTCTTATTTGCCAGA GTAGGAAAAGAGTAGATCTTTTTGCTCTCTAAATGTATTGATGGGTTGTGTTTTTTTTCCCACCTGCT AATAAATATTACATTGCAACATTCTTCCCTCAACTTCAAAACTGCTGAACTGAAACAATATGCATAAA AGAAAATCCTTTGCAGAAGAAAAAAAGCTATTTTCTCCCACTGATTTTGAATGGCACTTGCGGATGCA GTTCGCAAATCCTATTGCCTATTCCCTCATGAACATTGTGAAATGAAACCTTTGGACAGTCTGCCGCA TTGCGCATGAGACTGCCTGCGCAAGGCAAGGGTATGGTTCCCAAAGCACCCAGTGGTAAATCCTAAC TTATTATTCCCTTAAAATTCCAATGTAACAACGTGGGCCATAAAAGAGTTTCTGAACAAAACATGTCA CTTTGTGGAAAGGTGTTTTTCGTAATTAATGATGGAATCATGCTCATTTCAAAATGGAGGTCCACGAT TTGTGGCCAGCTGATGCCTGCAAATTATCCTGGATCACTAACTCTGA

SEQ ID:6 >STAR35

CGACTTGGTGATGCGGGCTCTTTTTTGGTTCCATATGAACTTTAAAGTAGTCTTTTCCAATTCTGTGA AGAAAGTCATTGGTAGGTTGATGGGGATGGCATTGAATCTGTAAATTACCTTGGGCAGTATGGCCAT TTTCACAATGTTGATTCTTCCTATCCATGATGATGGAATGTTCTTCCATTAGTTTGTATCCTCTTTTAT TTCCTTGAGCAGTGGTTTGTAGTTCTCCTTGAAGAGGTCCTTCACATCCCTTGTAAGTTGGATTCCTA GGTATTTTATTCTCTTTGAAGCAATTGTGAATGGGAGTTCACTCACGATTTGGCTCTCTGTTTGTCTG CTGGTGTATAAGAATGTTTGTGATTTTTGTACATTGATTTTGTATCCTGAGACTTTGCTGAAGTTGCT TATCAGCTTAAGGAGCTTTTGGGCTGAGACAATGGGATTTTCTAGATATACAATCATGTCGTCTGCAA ACAGGGACAATTTGACTTCCTCTTTTCCTAATTGAATACACTTTATCTCCTTCTCCTGCCTAATTGCCC TGGGCAGAACTTCCAACACTATGTTGAATAGGAGTGGTGAGAGAGGGCATCCCTGTCTTGTGCCAGT TTTCAAAGGGAATGCTTCCAGTTTTTGCCCATTCAGTATGATATTGGCTGTGGGTTTGTCATAGATAG CTCTTATTATTTTGAAATGTGTCCCATCAATACCTAATTTATTGAGAGTTTTTAGCATGAAGCATTGTT GAATTTTGTCAAAGGCTTTTTCTGCATCTATTGAGATAATCATGTGGTTTTTGTCTTTGGCTCTGTTTA TATGCTGGATTACATTTATTGATTTGTGTATATTGAACCAGCCTTGCATCCCAGGGATGAAGCCCACT TGATC

SEQ ID: 7

>STAR40

GATCAAGAAAGCACTCCGGGCTCCAGAAGGAGCCTTCCAGGCCAGCTTTGAGCATAAGCTGCTGATG

AGCAGTGAGTGTCTTGAGTAGTGTTCAGGGCAGCATGTTACCATTCATGCTTGACTTCTAGCCAGTG TGACGAGAGGCTGGAGTCAGGTCTCTAGAGAGTTGAGCAGCTCCAGCCTTAGATCTCCCAGTCTTAT GCGGTGTGCCCATTCGCTTTGTGTCTGCAGTCCCCTGGCCACACCCAGTAACAGTTCTGGGATCTAT GGGAGTAGCTTCCTTAGTGAGCTTTCCCTTCAAATACTTTGCAACCAGGTAGAGAAGTTTGGAGTGA AGGTTTTGTTCTTCGTTTCTTCACAATATGGATATGCATCTTCTTTTGAAAATGTTAAAGTAAATTACC TCTCTTTTCAGATACTGTCTTCATGCGAACTTGGTATCCTGTTTCCATCCCAGCCTTCTATAACCCAG TAACATCTTTTTTGAAACCAGTGGGTGAGAAAGACACCTGGTCAGGAACGCGGACCACAGGACAACT CAGGCTCACCCACGGCATCAGACTAAAGGCAAACAAGGACTCTGTATAAAGTACCGGTGGCATGTGT ATTAGTGGAGATGCAGCCTGTGCTCTGCAGACAGGGAGTCACACAGACACTTTTCTATAATTTCTTA AGTGCTTTGAATGTTCAAGTAGAAAGTCTAACATTAAATTTGATTGAACAATTGTATATTCATGGAAT ATTTTGGAACGGAATACCAAAAAATGGCAATAGTGGTTCTTTCTGGATGGAAGACAAACTTTTCTTCT TTAAAATAAATTTTATTTTATATATTTGAGGTTGACCACATGACCTTAAGGATACATATAGACAGTAA ACTGGTTACTACAGTGAAGCAAATTAACATATCTACCATCGTACATAGTTACATTTTTTTGTGTGACA GGAACAGCTAAAATCTACGTATTTAACAAAACTCCTAAAGACAATACATTTTTATTAACTATAGCCCT CATGATGTACATTAGATC.

Table 2

Preferred TRAP sequences.

>lambda fragment 35711-38103

5'—

AGATCTGAATTGCTATGTTTAGTGAGTTGTATCTATTTATTTTTCAATAAA TACAATTGGTTAT GTGTTTTGGGGGCGATCGTGAGGCAAAGAAAACCCGGCGCTGAGGCCGG GTTATTCTTGTTCTCTGGTCAAATTATATAGTTGGAAAACAAGGATGCATA TATGAATGAACGATGCAGAGGCAATGCCGATGGCGATAGTGGGTATCATG TAGCCGCTTATGCTGGAAAGAAGCAATAACCCGCAGAAAAACAAAGCTCC AAGCTCAACAAAACTAAGGGCATAGACAATAACTACCGATGTCATATACC CATACTCTCTAATCTTGGCCAGTCGGCGCGTTCTGCTTCCGATTAGAAACG TCAAGGCAGCAATCAGGATTGCAATCATGGTTCCTGCATATGATGACAAT GTCGCCCCAAGACCATCTCTATGAGCTGAAAAAGAAACACCAGGAATGTA GTGGCGGAAAAGGAGATAGCAAATGCTTACGATAACGTAAGGAATTATTA CTATGTAAACACCAGGCATGATTCTGTTCCGCATAATTACTCCTGATAATT AATCCTTAACTTTGCCCACCTGCCTTTTAAAACATTCCAGTATATCACTTTT CATTCTTGCGTAGCAATATGCCATCTCTTCAGCTATCTCAGCATTGGTGAC CTTGTTCAGAGGCGCTGAGAGATGGCCTTTTTCTGATAGATAATGTTCTGT TAAAATATCTCCGGCCTCATCTTTTGCCCGCAGGCTAATGTCTGAAAATTG AGGTGACGGGTTAAAAATAATATCCTTGGCAACCTTTTTTATATCCCTTTT AAATTTTGGCTTAATGACTATATCCAATGAGTCAAAAAGCTCCCCTTCAAT ATCTGTTGCCCCTAAGACCTTTAATATATCGCCAAATACAGGTAGCTTGGC TTCTACCTTCACCGTTGTTCGGCCGATGAAATGCATATGCATAACATCGTC TTTGGTGGTTCCCCTCATCAGTGGCTCTATCTGAACGCGCTCTCCACTGCT TAATGACATTCCTTTCCCGATTAAAAAATCTGTCAGATCGGATGTGGTCGG CCCGAAAACAGTTCTGGCAAAACCAATGGTGTCGCCTTCAACAAACAAAA AAGATGGGAATCCCAATGATTCGTCATCTGCGAGGCTGTTCTTAATATCTT CAACTGAAGCTTTAGAGCGATTTATCTTCTGAACCAGACTCTTGTCATTTG TTTTGGTAAAGAGAAAAGTTTTTCCATCGATTTTATGAATATACAAATAAT TGGAGCCAACCTGCAGGTGATGATTATCAGCCAGCAGAGAATTAAGGAAA ACAGACAGGTTTATTGAGCGCTTATCTTTCCCTTTATTTTTGCTGCGGTAA GTCGCATAAAAACCATTCTTCATAATTCAATCCATTTACTATGTTATGTTC TGAGGGGAGTGAAAATTCCCCTAATTCGATGAAGATTCTTGCTCAATTGTT ATCAGCTATGCGCCGACCAGAACACCTTGCCGATCAGCCAAACGTCTCTT CAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTG CATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACC GCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGC TATGCAGAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTA ACATTCCGTCAGGAAAGCTTGGCTTGGAGCCTGTTGGTGCGGTCATGGAA TTACCTTCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTG CTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCAGGTGAG AACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTA AGTGACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCG ATTGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAAT GCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAAC GCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGC CAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCT CAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCATACGTTAAATCT ATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCT CTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGGAACAACGC ATAACCCTGAAAGATTATGCAATGCGCTTTGGGCAAACCAAGACAGCTAA AGATCT— 3'

>Lambda fragment 22425-27972 5'-

CTGCAGATCTGGAAATTGCAACGAAGGAAGAAACCTCGTTGCTGGAAGCCTGGAAGAAGTATCGG GTGTTGCTGAACCGTGTTGATACATCAACTGCACCTGATATTGAGTGGCCTGCTGTCCCTGTTATG GAGTAATCGTTTTGTGATATGCCGCAGAAACGTTGTATGAAATAACGTTCTGCGGTTAGTTAGTAT

ATTGTAAAGCTGAGTATTGGTTTATTTGGCGATTATTATCTTCAGGAGAATAATGGAAGTTCTATGA CTCAATTGTTCATAGTGTTTACATCACCGCCAATTGCTTTTAAGACTGAACGCATGAAATATGGTTT TTCGTCATGTTTTGAGTCTGCTGTTGATATTTCTAAAGTCGGTTTTTTTTCTTCGTTTTCTCTAACTA TTTTCCATGAAATACATTTTTGATTATTATTTGAATCAATTCCAATTACCTGAAGTCTTTCATCTATA ATTGGCATTGTATGTATTGGTTTATTGGAGTAGATGCTTGCTTTTCTGAGCCATAGCTCTGATATCC AAATGAAGCCATAGGCATTTGTTATTTTGGCTCTGTCAGCTGCATAACGCCAAAAAATATATTTATC TGCTTGATCTTCAAATGTTGTATTGATTAAATCAATTGGATGGAATTGTTTATCATAAAAAATTAAT

GTTTGAATGTGATAACCGTCCTTTAAAAAAGTCGTTTCTGCAAGCTTGGCTGTATAGTCAACTAACT CTTCTGTCGAAGTGATATTTTTAGGCTTATCTACCAGTTTTAGACGCTCTTTAATATCTTCAGGAAT TATTTTATTGTCATATTGTATCATGCTAAATGACAATTTGCTTATGGAGTAATCTTTTAATTTTAAAT AAGTTATTCTCCTGGCTTCATCAAATAAAGAGTCGAATGATGTTGGCGAAATCACATCGTCACCCAT TGGATTGTTTATTTGTATGCCAAGAGAGTTACAGCAGTTATACATTCTGCCATAGATTATAGCTAAG

GCATGTAATAATTCGTAATCTTTTAGCGTATTAGCGACCCATCGTCTTTCTGATTTAATAATAGATG ATTCAGTTAAATATGAAGGTAATTTCTTTTGTGCAAGTCTGACTAACTTTTTTATACCAATGTTTAAC ATACTTTCATTTGTAATAAACTCAATGTCATTTTCTTCAATGTAAGATGAAATAAGAGTAGCCTTTG CCTCGCTATACATTTCTAAATCGCCTTGTTTTTCTATCGTATTGCGAGAATTTTTAGCCCAAGCCAT TAATGGATCATTTTTCCATTTTTCAATAACATTATTGTTATACCAAATGTCATATCCTATAATCTGGT

TTTTGTTTTTTTGAATAATAAATGTTACTGTTCTTGCGGTTTGGAGGAATTGATTCAAATTCAAGCG AAATAATTCAGGGTCAAAATATGTATCAATGCAGCATTTGAGCAAGTGCGATAAATCTTTAAGTCTT CTTTCCCATGGTTTTTTAGTCATAAAACTCTCCATTTTGATAGGTTGCATGCTAGATGCTGATATAT TTTAGAGGTGATAAAATTAACTGCTTAACTGTCAATGTAATACAAGTTGTTTGATCTTTGCAATGAT TCTTATCAGAAACCATATAGTAAATTAGTTACACAGGAAATTTTTAATATTATTATTATCATTCATTA

TGTATTAAAATTAGAGTTGTGGCTTGGCTCTGCTAACACGTTGCTCATAGGAGATATGGTAGAGCC GCAGACACGTCGTATGCAGGAACGTGCTGCGGCTGGCTGGTGAACTTCCGATAGTGCGGGTGTTG AATGATTTCCAGTTGCTACCGATTTTACATATTTTTTGCATGAGAGAATTTGTACCACCTCCCACCG ACCATCTATGACTGTACGCCACTGTCCCTAGGACTGCTATGTGCCGGAGCGGACATTACAAACGTC CTTCTCGGTGCATGCCACTGTTGCCAATGACCTGCCTAGGAATTGGTTAGCAAGTTACTACCGGAT

TTTGTAAAAACAGCCCTCCTCATATAAAAAGTATTCGTTCACTTCCGATAAGCGTCGTAATTTTCTA TCTTTCATCATATTCTAGATCCCTCTGAAAAAATCTTCCGAGTTTGCTAGGCACTGATACATAACTC TTTTCCAATAATTGGGGAAGTCATTCAAATCTATAATAGGTTTCAGATTTGCTTCAATAAATTCTGA CTGTAGCTGCTGAAACGTTGCGGTTGAACTATATTTCCTTATAACTTTTACGAAAGAGTTTCTTTGA GTAATCACTTCACTCAAGTGCTTCCCTGCCTCCAAACGATACCTGTTAGCAATATTTAATAGCTTGA

AATGATGAAGAGCTCTGTGTTTGTCTTCCTGCCTCCAGTTCGCCGGGCATTCAACATAAAAACTGA TAGCACCCGGAGTTCCGGAAACGAAATTTGCATATACCCATTGCTCACGAAAAAAAATGTCCTTGT CGATATAGGGATGAATCGCTTGGTGTACCTCATCTACTGCGAAAACTTGACCTTTCTCTCCCATATT GCAGTCGCGGCACGATGGAACTAAATTAATAGGCATCACCGAAAATTCAGGATAATGTGCAATAGG AAGAAAATGATCTATATTTTTTGTCTGTCCTATATCACCACAAAATGGACATTTTTCACCTGATGAA

ACAAGCATGTCATCGTAATATGTTCTAGCGGGTTTGTTTTTATCTCGGAGATTATTTTCATAAAGCT TTTCTAATTTAACCTTTGTCAGGTTACCAACTACTAAGGTTGTAGGCTCAAGAGGGTGTGTCCTGTC GTAGGTAAATAACTGACCTGTCGAGCTTAATATTCTATATTGTTGTTCTTTCTGCAAAAAAGTGGGG AAGTGAGTAATGAAATTATTTCTAACATTTATCTGCATCATACCTTCCGAGCATTTATTAAGCATTT CGCTATAAGTTCTCGCTGGAAGAGGTAGTTTTTTCATTGTACTTTACCTTCATCTGTGTTCATTATC ATCGCTTTTAAAACGGTTCGACCTTCTAATCCTATCTGACCATTATAATTTTTTAGAATGGTTTCATA AGAAAGCTCTGAATCAACGGAGTGCGATAATAAGTGGTGGTATGCAGAATTTGTCACTTCAAGTAA AAACACCTCACGAGTTAAAACACCTAAGTTCTCACCGAATGTCTCAATATCCGGACGGATAATATTT ATTGCTTCTCTTGACCGTAGGACTTTCCACATGCAGGATTTTGGAACCTCTTGCAGTACTACTGGG GAATGAGTTGCAATTATTGCTACACCATTGCGTGCATCGAGTAAGTCGCTTAATGTTCGTAAAAAA

GCAGAGAGCAAAGGTGGATGCAGATGAACCTCTGGTTCATCGAATAAAACTAATGACTTTTCGCCA ACGACATCTACTAATCTTGTGATAGTAAATAAAACAATTGCATGTCCAGAGCTCATTCGAAGCAGAT ATTTCTGGATATTGTCATAAAACAATTTAGTGAATTTATCATCGTCCACTTGAATCTGTGGTTCATT ACGTCTTAACTCTTCATATTTAGAAATGAGGCTGATGAGTTCCATATTTGAAAAGTTTTCATCACTA CTTAGTTTTTTGATAGCTTCAAGCCAGAGTTGTCTTTTTCTATCTACTCTCATACAACCAATAAATG

CTGAAATGAATTCTAAGCGGAGATCGCCTAGTGATTTTAAACTATTGCTGGCAGCATTCTTGAGTC CAATATAAAAGTATTGTGTACCTTTTGCTGGGTCAGGTTGTTCTTTAGGAGGAGTAAAAGGATCAA ATGCACTAAACGAAACTGAAACAAGCGATCGAAAATATCCCTTTGGGATTCTTGACTCGATAAGTC TATTATTTTCAGAGAAAAAATATTCATTGTTTTCTGGGTTGGTGATTGCACCAATCATTCCATTCAA AATTGTTGTTTTACCACACCCATTCCGCCCGATAAAAGCATGAATGTTCGTGCTGGGCATAGAATTA

ACCGTCACCTCAAAAGGTATAGTTAAATCACTGAATCCGGGAGCACTTTTTCTATTAAATGAAAAGT GGAAATCTGACAATTCTGGCAAACCATTTAACACACGTGCGAACTGTCCATGAATTTCTGAAAGAG TTACCCCTCTAAGTAATGAGGTGTTAAGGACGCTTTCATTTTCAATGTCGGCTAATCGATTTGGCCA TACTACTAAATCCTGAATAGCTTTAAGAAGGTTATGTTTAAAACCATCGCTTAATTTGCTGAGATTA ACATAGTAGTCAATGCTTTCACCTAAGGAAAAAAACATTTCAGGGAGTTGACTGAATTTTTTATCTA

TTAATGAATAAGTGCTTACTTCTTCTTTTTGACCTACAAAACCAATTTTAACATTTCCGATATCGCAT TTTTCACCATGCTCATCAAAGACAGTAAGATAAAACATTGTAACAAAGGAATAGTCATTCCAACCAT CTGCTCGTAGGAATGCCTTATTTTTTTCTACTGCAGGAATATACCCGCCTCTTTCAATAACACTAAA CTCCAACATATAGTAACCCTTAATTTTATTAAAATAACCGCAATTTATTTGGCGGCAACACAGGATC TCTCTTTTAAGTTACTCTCTATTACATACGTTTTCCATCTAAAAATTAGTAGTATTGAACTTAACGGG

GCATCGTATTGTAGTTTTCCATATTTAGCTTTCTGCTTCCTTTTGGATAACCCACTGTTATTCATGTT GCATGGTGCACTGTTTATACCAACGATATAGTCTATTAATGCATATATAGTATCGCCGAACGATTAG CTCTTCAGGCTTCTGAAGAAGCGTTTCAAGTACTAATAAGCCGATAGATAGCCACGGACTTCGTAG CCATTTTTCATAAGTGTTAACTTCCGCTCCTCGCTCATAACAGACATTCACTACAGTTATGGCGGAA AGGTATGCATGCTGGGTGTGGGGAAGTCGTGAAAGAAAAGAAGTCAGCTGCGTCGTTTGACATCA

CTGCTATCTTCTTACTGGTTATGCAGGTCGTAGTGGGTGGCACACAAAGCTTTGCACTGGATTGCG AGGCTTTGTGCTTCTCTGGAGTGCGACAGGTTTGATGACAAAAAATTAGCGCAAGAAGACAAAAAT CACCTTGCGCTAATGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCA TATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTT ATATCATTTTACGTTTCTCGTTCAGCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTTATC

AATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGATTTCAATTTTGTCCC ACTCCCTGCCTCTGTCATCACGATACTGTGATGCCATGGTGTCCGACTTATGCCCGAGAAGATGTT GAGCAAACTTATCGCTTATCTGCTTCTCATAGAGTCTTGCAGACAAACTGCGCAACTCGTGAAAGG TAGGCGGATCC— 3' >A combined synthetic polyA (SPA) sequence (version 1) and a pausing signal from the human α2 globin gene

5'— ATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTG AATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCC TGCAAGTGCAGGTGCCAGAACATTTCTCT— 3'

>A combined synthetic polyA (SPA) sequence (version 2) and a pausing signal from the human α2 globin gene 5'— AATAAAAGATCCTTATTTTCACTAGTTCTGTGTGTTGGTTTTTTGTGTG

AATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCC TGCAAGTGCAGGTGCCAGAACATTTCTCT— 3'

> Inter histone H3FA-H4F (http://genome.uesc.edu/cgi- bin/hgTraeks?hgsid=13148179&position=chr6%3A26063) (Chromosome 6; bp 26063887-26064766)

5'— ATTTGAGGACACTAACCTGTGCGCCATCCACGCCAAGCGCGTCACTATCATGCCCAAGGACAT CCAGCTCGCCCGCCGCATCCGCGGAGAGAGGGCGTGATTACTGTGGTCTCTCTGACGGTCCAAGCA AAGGCTCTTTTCAGAGCCACCACCTTTTCAAGTAAAGTAGCTGTAAGAAACCAATTTAAGACAAAAG GGAATGCATTGGGAGCACTTTTCGTTTTAATGCTACTGAAGGCTTCAAAACCAATCGATTTCGGCCG GTCGCGGTGACTCACGCCTGTAATTCAAGCACTTTGAGAGGCTGAGGCGGGCGGATTACCAGAAATC AGGAGTTCGGGATCAGCCTGGCCAACATGGCCGAATCCCGTCTCTACGAAAAATACAAAAACACGCC GGGCGCGACGGCGAGCGCTTGTAATCCCAGCTACACTCTGAAGGCTGAGGCAGGAGAAACACTTGA ACCTGAGAGGCAGAGGTTTCAGTGAATCGAGATGGCTCTAATGTACTCCAGTCTGGGCGACAGAGA GATTCGGTTAAAAAAAAAGTTCGACTTAAAATAATTCTGGAGTCAGAATGGGTTTACATTTAATTCTT AACCCAGTTCCTCAAAGCCTGTAGCTCTGTTAAGAAAATAAAGGCCATTGGTCAAGCCTGCTTGGTC CCACCCTCATCTCCCCACCCTCCCCCAATCGCTGCTCCCGCCATTTCCTGGGGCTTGGAGGAGGGGT TAAAGGAGCGGACTGTAGGCGTCACATTTCCCGCCTGCGCGCTTTTCAGTCTCAGTGTCCGCTGGAG GTGGGGGCAGGGGTAACGTAGATATATAAAGATCGGTTTCCTATTCTCTCACTTGCTCTTGGTTCAC TTCT— 3'

>Inter histone H1F4-H2BFB (chr6:26214737-26215909)

5^*— AAGGCGCCCAAGAGCCCAGCGAAGGCCAAAGCAGTTAAACCCAAGGCGGCTAAACCAAAGAC CGCCAAGCCCAAGGCAGCCAAGCCAAAGAAGGCGGCAGCCAAGAAAAAGTAGAAAGTTCCTTTGGC CAACTGCTTAGAAGCCCAACACAACCCAAAGGCTCTTTTCAGAGCCACCCACCGCTCTCAGTAAAAG AGCTGTTGCACTATTAGGGGGCGTGGCTCGGGAAAACGCTGCTAAGCAGGGGCGGGTCTCCCGGGA ACAAAGTCGGGGAGAGGAGTGGGATTTTGTGTGTCTCCGGAGCTATTTTTGACTAAGGCGTCGCGTC GCCCAAGCCGGAGTGCAGTGGCGTCATCTCGATTTTGCGTTCTCGAGTGTCGGAGTTGAACCCATTT GGGCCTCCCTTGTGCTTTGCACTTTTAGCAGGCCCTGGCCTCCAGATAGCATGGGAAAAAAAATGTT GGGATTTTCCCGGGTTTCTAAGCTGGGTTTTTCCGAGTTCCAAACACGGCACAGTGTATCAGTTTCT GTGCTGGTTACAAGCCTACTGGTTATCCCTATCGAGTATGGCAGGCAGTGAGGGACTTCAGAGGAGT ACGTCTTAGGACAAGTGGCATAGTACTGACATTATTTCCGAAGGGCTACATTTCAAGTGCTTGGGGA GACTACTGCCACATAACTGAAAATTAGAAACCGACACTGCAGAAAAATACTTGGTCCTTAAATGTGG CATTTGGATGGATTAAGGACTTGCCGAAACGTAAAACTGACAGACTTGGGGGGGGGGGATGTCCCA ATTAGCACGGCTTCTGTATGCAACGAGTCCCATACTTTGTTAAAGGAAGAAAGGAATGTGAGTTCTC CTAATCTGTTAAGTATCTTTCGGTGTAAGTTCTGACACCACAATGTTAAAAAAGTCGGATCTCAAAAA CCAACTGCTCCAAGCGAAGTGCACAGCTGTCTTGCCTAAAGAGGCCTATTTATAGTAGCCTCGGGTA GTCTGGTCTGGGCTTTCTCATTGGGTACAAGTAAAGGAACGAAATAGCCAATGAAAAGGTAGACTTT TAAGTGTCGTTTACATTGGCATTTGTGACGACACTCTAAAATTAATCCAATCATAAACGAAATCTGAT TAACCTCATTTGAATACCGCATCTATAAATGAACAGGGCC-- 3'

Claims

1. A method for providing a ceU with a protein expression unit comprising providing a nucleic acid comprising said unit with a nucleic acid encoding a binding site for a member of a chromatin modification system for rendering chromatin more accessible for transcription (opener), wherein said opener is present in said ceU, said method further comprising providing said expression unit to said ceU and culturing said ceU to aUow expression of said protein expression unit.

2. A method according to claim 1, wherein said opener comprises a histone modification enzyme.

3. A method according to claim 1 or claim 2, wherein said histone modifcation enzyme comprises a histone-acetyltransferase or a histone methyltransferase .

4. A method according to claim 3, wherein said histone- acetyltransferase comprises a p300/CBP protein, a P/CAF protein CBP, or a functional part, derivative and/or analogue thereof.

5. A method according to claim 3, wherein said histone methyltransferase comprises an Ashl protein or a functional part, derivative and/or analogue thereof.

6. A method according to claim 1, wherein said opener comprises a chromatin-remodelHng protein.

7. A method according to claim 6, wherein said chromatin-remodelHng protein comprises a trithorax group protein, a CHRAC group protein, a ACF group protein, a NURF group protein or a functional part, derivative and/or analogue thereof.

8. A method according to claim 7, wherein said opener comprises an

ISWI protein or a functional part, derivative and/or analogue thereof.

9. A method according to claim 1, wherein said opener comprises a trithorax group protein or a functional part , derivative and/or analogue thereof.

10. A method according to claim 9, wherein said trithorax group protein comprises a trithorax protein, a trithorax-like protein, , an ISWI protein, an

Ash protein, a moira protein, an osa protein or a functional part, derivative and/or analogue thereof.

11. A method according to any one of claims 1-10, wherein said protein expression unit comprises a promoter that is normaUy transcriptionally active in said cell.

12. A method according to any one of claims 1-11, wherein said opener is provided to the ceU.

13. A method according to claim 12, wherein said opener is a fusion protein comprising at least a functional part of an opener according to any one of claims 1-12, and a sequence specific nucleic acid binding domain.

14. A method according to claim 13, wherein said sequence specific nucleic acid binding domain is derived from a zinc-finger protein, a bacterial DNA binding protein, a yeast or fungus DNA binding protein.

15. A method according to claim 14, wherein said DNA binding protein is LexA or Gal4 or functional part, derivative and/or analogue thereof.

16. A method according to any one of claims 1-15, wherein said protein expression unit comprising said nucleic acid binding site for said opener is flanked on one or both sides by a nucleic acid comprising a STabiHzing Anti- Repressor (STAR) sequence.

17. A method according to claim 16, wherein said STAR sequence comprises a sequence as depicted in table I or a functional part, derivative and/or analogue thereof.

18. A method according to any one of claims 1-17, wherein said protein expression unit further comprises at least one TRAnscription Pause (TRAP) sequence and wherein said TRAP sequence is located downstream of an open reading frame in said expression unit in an orientation that can at least in part prevent the formation of anti-sense RNA.

19. A method according to any one of claims 1-18, wherein said protein expression unit further comprises at least one TRAnscription Pause (TRAP) sequence and wherein said TRAP sequence is located, (i) upstream of the combination of an open reading frame and a promoter driving transcription thereof in said expression unit, and (n) present in an orientation that can at least in part prevent transcription to enter said protein expression unit.

20. A method according to claim 18 or claim 19, wherein said protein expression unit comprises at least two TRAP sequences.

21. A method according to claim 20, wherein said at least two TRAP sequences are arranged such that the combination of an open reading frame and a promoter driving transcription thereof is flanked on both sides by at least one TRAP sequence.

22. A method according to any one of claims 18-21, wherein said protein expression unit is flanked by at least one TRAP sequence and at least one STAR sequence.

23. A method according to any one of claim 18-22, wherein said TRAP comprises an SPA/pause sequence or a functional part, derivative and/or analogue thereof.

24. A method according to claims 16-23, wherein two TRAP sequences flank a STAR element such that transcription is prevented transcription to enter said STAR element.

25. A method according to any one of claims 1-24, further comprising providing said ceU with a second protein expression unit.

26. A method according to any one of claims 1-25, wherein said protein expression unit comprises a multicistronic gene.

27. A method according to any one of claims 1-26, wherein said expression unit encodes a member of a multimeric protein.

28. A method according to any one of claims 1-27, wherein said protein expression unit encodes an immunoglobulin heavy or Hght chain or a antigen binding part, derivative and/or analogue thereof.

29. A method according to any one of claims 1-28, wherein said binding site is provided to the expression unit upstream of a promoter therein

30. A method according to any one of claims 1-29, wherein a promoter in said expression unit comprises a human cytomegalovirus, a simian virus 40, an ubiquitine C, an elongation factor one-alpha promoter or a functional part, derivative, analogue or combination thereof.

31. A method according to any one of claims 1-30, further comprising harvesting from said cell or culture supernatant thereof, a protein produced by said expression unit.

32. A ceU obtainable by a method according to any one of claims 1-31.

33. A cell according to claim 32, wherein said ceU is a vertebrate ceU or a plant ceU.

34. A ceU according to claim 33, wherein said ceU is a mammalian ceU, preferably a human ceU.

35. A cell according to claim 34, wherein said ceU comprises a U_ 2 OS osteosarcoma, CHO, 293, HuNS-1 myeloma, WERI-Rb-1 retinoblastoma, BHK, Vero, non-secreting mouse myeloma Sp2/0-Ag 14, non-secreting mouse myeloma NSO, or NCI-H295R adrenal gland carcinoma cell.

36. A nucleic acid comprising a protein expression unit which is provided with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener).

37. A nucleic acid according to claim 36, further comprising a STAR sequence.

38. A nucleic acid according to claim 37, further comprising a TRAP sequence either upstream of the combination of an open reading frame and a promoter driving transcription thereof in said protein expression unit in an orientation that at least in part prevents transcription from entering said protein expression unit or downstream of said open reading frame in an orientation that can at least in part prevent the formation of anti-sense RNA.

39 A nucleic acid according to any one of claims 36-38, wherein said expression unit comprises a human cytomegalovirus, a simian virus 40, an ubiquitine C, an elongation factor one-alpha promoter or a functional part, derivative, analogue or combination thereof.

40. A nucleic acid according to any one of claims 36-39, wherein said protein expression unit comprises a bi- or multicistronic gene.

41. A nucleic acid according to claim 40, further comprising an internal ribosomal entry site.

42. Use of an opener for stabUizing expression of an expression unit.

43. Use of an opener for increasing the number of clones expressing a certain amount of protein after genetic modification.

44. Use of an opener for increasing transcript levels produced by an expression unit.

45. A method for improving a function of a protein expression unit comprising a STAR element comprising providing said protein expression unit with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener).

46. A method for improving a function of a STAR element in a protein expression unit comprising providing said protein expression unit with a binding site for a member of an chromatin modification system for rendering chromatin more accessible for transcription (opener).

47. A ceU provided with a nucleic acid encoding an opener provided with a novel sequence specific nucleic acid binding activity.

48. A ceU provided with a nucleic acid according to any one of claims 36- 41.