EP0368926A1 - Cellules transfectees contenant des plasmides comportant des genes orientes dans des directions opposees et leur procede d'obtention - Google Patents

Cellules transfectees contenant des plasmides comportant des genes orientes dans des directions opposees et leur procede d'obtention

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Publication number
EP0368926A1
EP0368926A1 EP88907466A EP88907466A EP0368926A1 EP 0368926 A1 EP0368926 A1 EP 0368926A1 EP 88907466 A EP88907466 A EP 88907466A EP 88907466 A EP88907466 A EP 88907466A EP 0368926 A1 EP0368926 A1 EP 0368926A1
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European Patent Office
Prior art keywords
gene
cells
expression
plasmid
cell line
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP88907466A
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German (de)
English (en)
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EP0368926A4 (en
Inventor
Linda M. Cashion
Kathi A. Begley
Wendy W. Colby
Michael John Morser
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Berlex Laboratories Inc
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Berlex Laboratories Inc
Codon
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Publication of EP0368926A1 publication Critical patent/EP0368926A1/fr
Publication of EP0368926A4 publication Critical patent/EP0368926A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • This invention relates generally to recombinant DNA techniques and to the expression of mammalian polypeptides in genetically engineered eukaryotic cells. Specifically, the invention relates to preferred plasmid constructs and methods that increase levels of expression of a cloned gene product. These preferred plasmids have selectable and nonselected gene cassettes adjacent to each other and oriented in the opposite direction for transcription. Such an orientation enhances overall levels of expression for the nonselected gene. Further, this invention relates to gene products expressed at these very high levels by the herein described method, and to the eukaryotic cells derived thereby.
  • the current methodology usually includes first selecting a population of cells containing the transfected plasmid on the basis of drug resistance. This population is then screened for the introduction of a non-selectable gene, either by assaying for the non- selectable gene product, or for the presence of the genetic material comprising the coding sequence of the non-selectable gene. This method allows for the stable introduction into cultured mammalian cells of any cloned gene, and the systematic isolation of such cells.
  • Gene amplification is proven to be a method frequently used to increase expression levels. Gene amplification is often induced by exposure of sensitive cells to stepwise increases in antifolates, such as methotrexate (MTX), in the growth medium. This amplification can yield cells which are resistant to high levels of MTX and have increased levels of production of other associated genes as well (European Patent Application Nos. 0117059, 00117060 and Kaufman et al. (1985) Mol. Cell. Biol. 5:1750-1759, 1985).
  • MTX methotrexate
  • mutant cells In general, increased production of foreign proteins in a host through gene amplification techniques has been primarily limited to the use of mutant hosts. This is often undesirable for a number of reasons, most notably, that the mutant cell host most suitable for production of the foreign protein is unavailable.
  • This invention provides for eukaryotic cell lines which express a desired gene product, comprising host cells transfected with at least one recombinant expression plasmid wherein the expression plasmid comprises a selectable gene cassette and a second gene cassette encoding the desired gene product with the priviso that the selectable gene cassette and the second gene cassette are disposed adjacent to each other in opposite and divergent transcriptional orientation.
  • the desired gene product of the cell lines described is expressed at higher levels relative to host cells transfected with identically situated plasmids having either the second gene alone or the selectable gene cassette and the second gene cassette oriented in the same direction.
  • Preferred selectable gene cassettes are those that, contain sequences that encode a protein selected from: the group consisting of dihydrofolate reductase, neomycin phosphotransferase, and hygromycin phosphotransferase.
  • the eukaryotic cell line may express desired gene products that are either selectable or nonr-selectable, and the desired gene products can either: be heterologous or endogenous to the host cell.
  • Preferred desired genes encode a human pharmaceutical selected from the group consisting of hormones, immunogens, anti-cancer agents, antibiotics, immunoglobins, anti-allergy agents, or thrombolytic preparations.
  • the preferred non-selectable gene encodes tissue plasminogen activator of the human variety.
  • the desired genes encoding t-PA can optionally, but preferrably, contain introns such as a cDNA/genomic hybrid gene.
  • Preferred t-PA expression vectors are pPA003, pPA509, and pPA202.
  • Figures 1a-d are diagramatic gene maps of plasmids pPA502, pPA518, pPA519, pPA524, pPA525, pPA208, pPa209, pPA206, pPA207, pPa202, pPA509, pPA510, pPA003, pPA118, pPA119, pPA122, pPA102, pPA017.
  • Figure 2 illustrates the steps leading to the construction of transfection plasmid pPA003.
  • a The DNA sequence of the amino terminal region of the t-PA gene extending from intron A through the coding region and 5' - untranslated region to intron A'.
  • the dashed line shows the structure and sequence of the amino terminal region of the t-PA chromosomal gene as it is fused to t-PA cDNA to generate a hybrid genomic DNA-cDNA gene.
  • b The Nar I fragment of pPA103 extending from intron A to a Nar I site in t-PA cDNA was inserted into pPA104. This allowed removal of a 3.3 Kb Bcll-Bgl II fragment containing the Bgl II site. This fragment was ligated into pneo5 resulting in pPA003, the genomic hybrid t-PA expression plasmid.
  • FIG. 4 An analysis of the expression of t- PA plasmids in C127 cells.
  • C127 cells were transfected with plasmids pPA518, pPA519, pPA524, and pPA525 as well as a control plasmid lacking the LTR promoters and then plated into 100 mm perti dishes at different densities.
  • the plates were overlaid with agarose containing fibrin and plasminogen as described in the text.
  • One week later the number of clearings was counted.
  • the graph is a plot: of the number of clearings verus the cell density.
  • Figure 5 Shows the production of t-PA by CHO cells transfected with pPA206 and pPA207. The medium from 11 clones was assayed for production.
  • This invention involves a series of molecular genetic manipulations that can be achieved in a variety of known ways.
  • Prototype vectors and cell lines have been deposited in accordance with the Budapest Treaty. Plasmids pPA003, pPA202, and pPA509 are maintained in an E. coli host and are illustrated in Figure 1. Plasmid pPA003 was transfected into the cell line CHL-1 and a stable transfectant, CHL-2 was isolated. These plasmids and cell lines were deposited pursuant to the Budapest Treaty with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 (USA), and given the accession numbers: pPA003 deposited on January 13, 1987 and assigned ATCC 67293; pPA202 deposited on
  • Maniatis Maniatis.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage S.L. and Caruthers, M.H. Tetrahedron Letts. 22 (20):1859-1862 (1981) using an automated synthesizer, as described in Needham- VanDevanter, D.H., et al., Nucleic Acids Res., 12:6159- 6168 (1984). Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J.D. and Regnier, F.E., J. Chrom., 255:137-149 (1983).
  • the sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam, A.M. and Gilbert, W., Grossman, L. and Moldave, D., eds., Academic Press, New York, Methods in Enzymology, 65:499-560 (1980). Alternatively, the sequence can be confirmed after the assembly of the oligonucleotide fragments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double- stranded templates of Wallace, R.B., et al., Gene, 16:21-26 (1981). This invention relates to cloning and use of expression vectors in eukaryotic cells.
  • the host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into aminal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, electroporation and micro-injection of the DNA directly into the cells.
  • Cloning vectors suitable for replication in prokaryotes or eukaryotes and containing transcription terminators useful for regulation of the expression of downstream structural proteins are described herein.
  • the disclosed vectors are comprised of expression cassettes containing at least one independent terminator sequence; sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors; and selection markers for both the prokaryote and eukaryote systems.
  • selectable markers In order to select the transformed bacteria, selectable markers must be incorporated into the cloning vectors. These markers permit the selection of bacterial colonies containing the vectors which one desires to replicate. Examples of selectable markers include for E. coli: genes specifying resistance to antibiotics, i.e., ampicillin, tetracycline, chloramphenicol, kanamycin, erythromycin, or genes conferring other types of selectable enzymatic activities such as b-galactosidase. There are numerous other markers both known and unknown which embody the above scientific principles, all of which would be useful as markers to detect those bacteria transformed with the vectors embraced by this invention.
  • suitable eukaryote markers In order to select the transfected eukaryotic cells, suitable eukaryote markers must be incorporated into the cloning vectors. These markers may permit selection of transfected cells by virtue of survival in an otherwise lethal environment utilizing the same principles described for the prokaryote markers or the selectable markers may visibly alter the host cells allowing for easy detection. A general overview of this art is found in P.J. Southern and Berg, P.J. Mol. App. Gen. 1: 327-41 (1982).
  • selectable markers include the dihydrofolate reductase gene (dhfr), an altered dhfr gene (dhfr-IV), the hygromycin B resistance gene (hmb), the neomycin phosphotransferase II gene (neo) and the adenosine deaminase gene for the human melanoma cell line RPMI 7932 (Bowes) cells, Chinese hamster cells, C127 murine cells and other higher eukaryotic calls; and the enzyme, b-galactosidase and the nuclear polyhedral virus from Autographa californica for insect cell lines from Spodoptera frugiperda and Bombyx mori; and for yeast, Lue-2, URA-3, Trp-1, and His-3 are known selectable markers (Gene 8:17-24, 1979). There are numerous other markers both known and unknown which embody the above scientific principles, all of which would be useful as markers to detect those eukaryotic cells transf
  • the desired structural gene might also operate as a selectable marker and eliminate the need for a separate selectable marker in eukaryotic cell hosts.
  • the preferred selectable markers are neomycin phosphotransferase (neo), dihyrofolate reductase (dhfr), an altered dihydrofolate reductase (dhfr-IV), and phosphotransferase (hmb).
  • neo neomycin phosphotransferase
  • dhfr dihyrofolate reductase
  • dhfr-IV dihydrofolate reductase
  • hmb phosphotransferase
  • hmb gene confers resistance to hygromycin B (Blochlinger, K., and Digglemann, H., Mol. Cell. Biol 4:2929-2931, 1984).
  • the selectable genes may be heterologous or endogenous to the host cell.
  • heterologous selectable genes would be neo in RPMI 7932 cells, CHO cells, or Vero cells, or hmb in RPMI 7932, CHO, or Vero cells.
  • the selectable gene may also be a gene that can be amplified. DHFR is an example of such a selectable/amplifiable gene.
  • the expression vectors may comprises two or more selectable genes. This allows selection of transfectants using first one selection medium, and then a second selection medium. The level of expression of the desired gene product increases with each selection procedure.
  • selectable gene it will be operably linked to one or more regulatory sequences, e.g., the gene will be placed in the expression vector adjacent to a promoter so that expression can occur.
  • the structural gene will have its own host cell compatable promoter.
  • the eukaryotic cell lines useful in the practice of the present invention are wild-type or auxotrophic, such choice is governed by the particular selection marker used in the transfection plasmid.
  • wild-type cells can be used with a dhfr selection marker although wild-type cells have an endogenous dhfr gene, but drug resistance selection markers require a host cell which is not resistant to that drug.
  • the cloning vectors must contain an origin of replication suitable for directing replication in prokaryotes.
  • the vectors For maintenance in eukaryotic hosts the vectors must either contain an ⁇ origin of replication usually of viral origin or have the capacity to integrate into the host genome.
  • origin of replications for prokaryotes E. coli replicons, which are the most closely studied, have origins of replication which are temperature dependent, permit high copy replication or those which constitutively sustain plasmid copies at only lower moderate levels. Examples of E. coli origins of replication are ColE1 ori, R1 ori R, or pSC101 ori.
  • the plasmids After transfection into eukaryotic cells, the plasmids will either integrate into the host's genome or remain extrachromosomally replicating. In the embodiment exemplified, the plasmids described integrate into the host cell. Examples of non-integrating vectors include those derivatives from the Epstein Bar virus. Yates et al., Nature, 313: 812-814 (1985). There are numerous origins of replication both known and unknown which embody the above scientific principles, all of which would be useful to maintain the plasmids within the prokaryotic and eukaryotic hosts transformed, transfected or infected with the vectors embraced by this invention.
  • E. EUKARYOTIC HOST CELLS Of the higher eukaryotic cell systems useful for the expression of desired proteins, there are numerous cell systems to select from.
  • Illustrative examples of mammalian cell lines include RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7 , C127 or MDCK cell lines .
  • Cells suitable for use in this invention are commercially available from the American Type Culture Collection.
  • Illustrative insect cell lines include Spodoptera frugiperda (fall Armyworm) and Bombyx mori (silkworm).
  • the disclosed embodiment makes use of CHL-1 cells. These are derived from RPMI 7932 cells, a readily available human cell line.
  • the CHL-1 line is improved over the parental RPMI 7932 cell line.
  • the CHL-1 cells unlike the parental cells, have been cured of mycoplasma contamination, which is an opportunistic organism that frequently contaminates cells in long term culture and interferes with large scale, i.e. commercial, use in a production scheme.
  • the CHL-1 RPMI 7932 cell derivative can grow at high cell density than the parental cell line.
  • CHL-1 cells are capable of growth to a density of 5 X 10 7 cells/ml; whereas the parental RPMI 7932 cells will grow to only 5 X 10 6 cells/ml under the same conditions.
  • the eukaryotic expression cassettes are sequences of DNA which are functionally capable of directing the expression of proteins in a eukaryotic host.
  • the cassettes are generically comprised of a functional promoter that permits the initiation of transcription, at least one structural gene and an appropriate 3' portion encoding the polyadenylation signal sequences necessary to terminate the transcription process.
  • An example of a terminator sequence is the polyadenylation sequence from SV40.
  • the cassettes may optionally contain a signal sequence in the 5' region of the structural gene that will direct post-translational processing of the structural gene.
  • the polypeptides can be any variety of proteins, such as enzymes, immunoglobulins, hormones, vaccines, receptors and the like.
  • proteins falling within the above categories include tissue plasminogen activator, Factor VIII:C, interferons, insulin, growth hormone, growth factors, erythropietin, interleukins 1, 2, and 3, and others, as new genes are cloned and expressed.
  • tissue plasminogen activator Factor VIII:C
  • interferons insulin, growth hormone, growth factors, erythropietin, interleukins 1, 2, and 3, and others, as new genes are cloned and expressed.
  • the entire structural gene for the naturally occurring protein need not be expressed, as fragments or subunits may be produced as desired.
  • the proteins will contain leader sequences, transmembrance sequences or the like, depending upon the particular ultimate utility.
  • the precise structural gene incorporated into the expression vector is not critical to the practice of the present invention.
  • the gene for tissue plasminogen activator has been used to describe the current invention.
  • the structural gene encode tissue plasminogen activator is not selectable by growth in any defined medium. T-PA production must be determined by assay of surviving transfectants.
  • Other suitable non-selectable genes include by are not limited to human growth hormone, bovine growth hormone, erythropoetin, IgE, calf chymosin, glycosylation inhibiting factor (GIF), and urokinase.
  • promoters useful for regulating the expression of heterologous proteins in mammalian or insect systems include but are not limited to the following: the retroviral long terminal repeat promoters (Nature, 297:479-483, 1982), SV-40 promoter
  • This invention relates to the expression of desired proteins in eukaryotic host cells. It is expected that those of skill in the art are knowledgeable in the expression systems chosen for ultimate expression of any chosen protein and no attempt to describe in detail the various methods known for the expression of proteins in eukaryotes will be made. However, several general references are available which describe in detail the processes for expression of proteins in eukaryotic cell systems. These references cite two additional reference which give even greater detail. For example, the expression of proteins in yeast is generally described in Methods In Yeast Genetics, Sherman, F., et al., Cold Spring Harbor
  • the host cell is capable of rapid growth in cell culture and able to glycosylate expressed gene products to ensure that the protein is produced in high quantity and resembles the naturally occurring material.
  • Cells known to be suitable for dense growth in tissue culture are particularly desirable and in the art a variety of invertebrate or vertebrate cells have been employed whether normal or transformed.
  • the transfected cells are grown up by means well known in the art. For examples, see: Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc. (1977).
  • the expression products are harvested from the cell medium in those systems where the protein is excreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or enzymatic means, which are well known in the art.
  • a eukaryotic host cell is transfected with at least one vector containing at least one selectable gene and a structural gene of interest.
  • Each gene is operably linked to a promoter which allows that gene to be transcribed into mRNA.
  • This promoter-gene cassette is transcribed unidirectionally; from the 5' portion of the gene, adjacent to the promoter, to the 3' portion of the gene adjacent to a terminator sequence.
  • plasmids are constructed wherein at least one selectable gene cassette is transcribed in the opposite and divergent direction as an adjacent structural gene of interest. In the divergent form the promoters of the selectable gene and the gene of interest are adjacent as diagrammed in Fig 3.
  • Transfectants are selected by growth under suitable selective conditions according to the selectable gene present on the expression vector. A transfected cell may then be isolated and characterized.
  • the structural gene of interest and the selectable genes are operably linked to one or more regulatory DNA sequences.
  • the resultant transfected cell line is then grown under appropriate conditions to allow for expression of the structural gene of interest, and followed by the subsequent purification and recovery of that structural gene product.
  • the disclosed methods provide plasmids wherein the adjacent promotors of the selectable gene cassette and the gene cassette encoding the desired protein are at most 2.0 kilobases apart. In the preferred embodiments the promoters of these adjacent gene cassettes are 100-1000 base pairs apart. Additionally, no other structural gene lies between the selectable gene and the gene of interest.
  • the plasmid encoded structural gene of the examples is endogenous to the host cell. It is also possible to practice the current invention to express proteins that are heterologous, that is, proteins whose DNA sequence is not normally present in the host cell. According to the present invention, plasmids have been prepared which can be transfected into eukaryotic cells by virtue of a selectable gene. The selectable gene allows eukaryotic cells transfected with the plasmids of the present invention to be selectively grown in the presence of an agent.
  • the general method used to isolate clones has been to introduce purified plasmid DNA into cells that had been plated 24 hours previously by the calcium phosphate precipitation technique (Wigler PNAS 76: 1376- 1376, 1979).
  • t-PA expression As a mass culture value. Individual clones were then isolated from the population to determine the distribution of expression levels of clones within the population. Numerous assays are available for determining the level of t-PA produced by cells in culture. These methods are described in detail in the following references: Kruithof, et al., Thromb. Res., 28:251-260; Verheijen, J.H., et al., Thromb. Res., 39:281-288, 1985; Beebe, et al., Thromb. Res., 47:123-128, 1987; and Gaffney, P., et al., Thromb. and Haemos, 53:134-136, 1985.
  • CHL-1 (A.T.C.C. Accession No. CRL 9446) is the cell line used for most of these experiments.
  • CHL-1 is a derivative of RPMI 7932 cells described in the detailed description.
  • a second cell line, CHL-2 (A.T.C.C. Accession No. CRL 9451) is derived from CHL-1 by a previous transfection with pPA003. This stably transfected cell line produces t-PA at a rate of 0.20-0.35 mU/cell/day; about 2-3 times more than the parent CHL-1.
  • both pPA524 and pPA525 were more efficient in generating colonies that produced detectable levels of t-PA than either pPA518 or pPA519. This result suggests that position of the selection marker does influence t-PA expression. Moreover, the number of clearings seen with pPA525 was greater than with pPA524 indicating that gene orientation in the divergent form has a positive effect on expression of t- PA. This example demonstrates that the enhanced differential t-PA expression due to gene orientation described in example F.i. is not restricted to the CHL-1 cell line.
  • Plasmids pPA208 and pPA209 were constructed so that the dhfr gene can be used as the selection marker in place of hygromycin. Plasmid pPA208 contains the dhfr gene and the t-PA genes in the same orientation; in pPA209 the genes are in the divergent orientation. These t-PA expression plasmids were analyzed to determine if the effect of gene orientation was specific for the hygromycin resistance gene or if other selection markers can be substituted.
  • This pair of plasmids was used to transfect CHL-1 cells and clones were selected in 100 nM MTX in medium lacking nucleosides.
  • pSC614, a dhfr plasmid without t-PA sequences, and salmon sperm DNA were used as transfection controls.
  • the transfection frequency and the t-PA production level of the mass cultures were determined.
  • Plasmid pPA209 yielded a transfection frequency ten-fold higher than did plasmid pPA208. Further, t-PA production in the mass culture was determined to be twice as high for the pPA209 transfectants than for the pPA208 transfectants (Table IIA).
  • T-PA levels for individual clones resulting from transfections into CHL-2 with pPA509 and pPA510 follow the same pattern (Table III C).
  • hygromycin was the selective agent
  • t-PA levels in clones from pPA509 and pPA510 were identical.
  • clones from the pPA509 transfection gave more clones and they had higher levels of t-PA (over 15 mu/c/d).
  • the increase in t-PA expression by the highest clones derived from CHL-2 is almost 5 times greater than the highest clone from CHL-1 (compare Table IIIC with IIIB). This larger increase is not expected from the production levels of the two host cell lines.
  • neo gene in both pPA206 and pPA207 is oriented in the same direction of transcription as the t-PA gene.
  • the t-PA expression in the mass culture showed little difference between the two plasmids.
  • the transcription orientation of the dhfr gene relative to the t-PA gene is in the same direction in pPA206 and in the divergent orientation in pPA207.
  • the selective agent was switched to 100nM MTX, the effect of the gene orientation is seen.
  • the determinations of t-PA production in the mass cultures show that for pPA207 transfectants, this level is four times greater (1.322 mU/cell/day) than that of pPA206 transfectants (0.322 mU/cell/day).
  • the range of clones expressing t-PA is shifted to higher levels for pPA207 transfectants. The highest clone was producing 6.7 mU/cell/day of t-PA.
  • CHO dhfr-cells were transfected with pPA206 and pPA207 plasmids and selected for growth in media lacking nucleosides (Kaufman, R., and Sharp, P., J. Mol. Biol., 159:601-621, 1982). Individual clones were then assayed for productivity ( Figure 5). The transfectants from pPA207 had more clones producing higher levels of t-PA in the initial screen and had a range of clones with greater levels of t-PA synthesis than pPA206 transfectants. The finding from this experiment is consistent with Examples F.iii. and v. in that the different orientation of the dhfr gene in pPA207 and pPA206 has an marked effect on t-PA production.
  • This example also demonstrates that the increased t-PA expression observed with plasmids having the selection marker in a divergent transcription orientation is not restricted to host cell or selection marker.
  • Examples F.i., ii., and vi. together show that the effect of gene orientation can be demonstrated in three different cell lines from human, mouse, and hamster. Moreover, this effect is seen when either hygromycin or dhfr is used as the selection marker.
  • t-PA expression levels of the clones described in Example F.iv. were increased by stepwise selection in MTX. Initially, a mass population was generated by transfection of CHL-1 cells with pPA509. This population of hygromycin resistant clones was then selected in 100nM MTX. The ma ⁇ .s culture of 100nM MTX resistant clones was then subjected to a second round of amplification in 1 ⁇ M MTX. From this selection procedure two new clones were isolated W8A5 and W8A6. They were propagated in the absence and presence of l ⁇ M MTX.
  • W8A5 without MTX grew very well and initially produced very high levels of t-PA (5 mU/c/d) (Table V). However, the expression level decreased with growth in the absence of selective pressure (down to 1.8 mU/c/d). This culture was then reselected in 1 ⁇ M MTX and yielded clones W8A5-1, and W8A5-3.
  • Clones W10A5, 11, 12, 17, 21 and 24 were derived by transfecting plasmid pPA509 into CHL-2 cells and selecting for hmb resistance (Example F.iv.). A mass population of hygromycin resistant clones were selected in 500nM MTX and expressed very high levels of t-PA. Individual clones were isolated from this population in 500nM MTX and the clones W10A5, W10A11, W10A12, W10A17, W10A21 and W10A24 were analyzed for t-PA expression.
  • plasmid pPA202 was used for a similar experiment.
  • This plasmid carries two selection markers, dhfr-IV and neo.
  • the dhfr-IV gene under the control of the SVe promoter was inserted 5' to the t-PA gene and is transcribed in a divergent direction with respect to t-PA.
  • the neo gene, under control of the TK promoter was inserted 3' to the t-PA gene and is transcribed convergent to t-PA. No paired plasmid with reverse orientation was constructed.
  • CHL-1 cells were transfected with pPA202 and transfectants were selected with neomycin.

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Abstract

Cette invention concerne des techniques recombinantes à base d'ADN ainsi que l'expression de poleptides mammifères dans des cellules eucaryotiques obtenues par génie génétique, plus précisément, elle se rapporte à des constructions de plasmides préférées et à des procédés améliorant les niveaux d'expression d'un produit génétique cloné. Ces plasmides préférés ont des cassettes génétiques sélectionnables et non sélectionnées, adjacentes les unes par rapport aux autres, et orientées dans la direction opposée pour transcription. Une telle orientation améliore les niveaux globaux d'expression pour le gène non sélectionné. En outre, cette invention a trait à des produits génétiques exprimés à ces niveaux très élevés par le procédé ici décrit, et à des cellules eucaryotiques ainsi dérivées.
EP19880907466 1987-07-16 1988-07-15 Transfected cells containing plasmids having genes oriented in opposing directions and methods of obtaining the same Withdrawn EP0368926A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7408387A 1987-07-16 1987-07-16
US74083 1993-06-08

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EP0368926A1 true EP0368926A1 (fr) 1990-05-23
EP0368926A4 EP0368926A4 (en) 1991-11-27

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AU (1) AU2263288A (fr)
WO (1) WO1989000605A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE3744595A1 (de) * 1987-12-31 1989-07-13 Andreas Dr Plueckthun Verfahren zur gentechnischen herstellung von antikoerpern
DE4410188A1 (de) * 1994-03-24 1995-09-28 Boehringer Mannheim Gmbh Gentherapeutisches Verfahren unter Verwendung von antibiotikaresistenzgenfreien DNA-Vektoren
GB9603803D0 (en) * 1996-02-22 1996-04-24 Zeneca Ltd Production of proteins, plasmids coding therefor and organisms containing such plasmids
EP1308517A1 (fr) * 2001-10-31 2003-05-07 Aventis Pharmacueticals Products Inc. Vecteurs pour l'expression de multiples transgenes
PT2592148T (pt) 2007-10-12 2018-11-12 Hoffmann La Roche Expressão proteica de múltiplos ácidos nucleicos
JP7021245B2 (ja) 2017-03-10 2022-02-16 エフ．ホフマン－ラ ロシュ アーゲー 多重特異性抗体を生産するための方法

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PT73614B (en) * 1980-09-11 1983-10-19 Merck & Co Inc Method for preparing mycoplasma-free hepatoma cell line
NZ206699A (en) * 1982-12-30 1989-08-29 Bio Response Inc Process for the production of serum independent cell lines
US4663281A (en) * 1984-03-22 1987-05-05 Mass Institute Of Technology Enhanced production of proteinaceous materials in eucaryotic cells
EP0192658A4 (fr) * 1984-07-30 1987-07-13 Salk Inst For Biological Studi Vecteurs retroviraux de transfert genetique.
EP0216846B2 (fr) * 1985-04-01 1995-04-26 Celltech Limited Lignee cellulaire de myelomes transformee et procede d'expression d'un gene codant un polypeptide eucaryotique employant cette lignee
AU593264B2 (en) * 1985-07-10 1990-02-08 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Chromosomal DNA sequence, expression vector for human tissue plasminogen activating factor, cultured cells transfected with same and method of producing said activating factor

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Publication number Publication date
EP0368926A4 (en) 1991-11-27
WO1989000605A1 (fr) 1989-01-26
AU2263288A (en) 1989-02-13

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