JPH0795880A - Cotransformed eukaryotic cells - Google Patents

Abstract

(57) [Summary] [Objective] The object of the present invention is to interferon, insulin, growth hormone, coagulation factor, viral antigen / antibody,
It mainly consists in efficiently producing proteinaceous products such as enzymes. [Structure] DNA encoding genetic information on target protein-like products such as interferon, insulin, growth hormone, coagulation factor, viral antigen / antibody, enzyme, etc.
Provided is a eukaryotic cell co-transformed with an A molecule and a DNA B molecule encoding genetic information that confers methotrexate resistance.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention introduces a DNA containing a genetic information substance, that is, a gene encoding a protein-like substance and / or a gene that regulates or otherwise affects the production of the protein-like substance. Cells of organisms classified as superfauna and eukaryotes, including those of the plant and animal kingdoms. Such genetic interference is commonly referred to as genetic engineering, and in some respect recombinant DNA.
It includes the use of technology. The disclosed invention is particularly
It should be distinguished from the cells of prokaryotic plants and animals that contain DNA-transferred bacteria. This distinction is based in terms of fundamental differences between eukaryotic and prokaryotic cells, the former characterized by the eukaryote formed by the envelope and meiosis and the latter by the absence of well-defined nuclei and Characterized by the absence of meiosis. Furthermore, at the genetic level, many genes in eukaryotes are disrupted by contiguous, non-collinear, non-coding linkages, whereas in prokaryotes, genes are contiguous and co-linear.

[0002]

BACKGROUND OF THE INVENTION Although the understanding of prokaryotes, in particular bacteria, relevant to most part has progressed independently of advances in the understanding of eukaryotes, it is the present invention to describe certain developments involving prokaryotes. Will be helpful to the evaluation of. In 1944, Avery was the DN of the cellular gene
We reported transformation of prokaryotic cells using A-mediated transfer (Av
ery, OT et al., J. Exp. Med. 79: 137-158 (1944)).
Subsequently, reports of transformed prokaryotic cells appeared in the literature. In 1975, Cohen et al. Reported results involving a first transformation and then cotransformation of the prokaryotic Escherichia coli (Kretschmer, PJ et al., J. Bacteriology.
124: 225-231 (1955)). In the experiments reported therein, the authors disclosed co-transformation of prokaryotic cells with naturally occurring plasmid DNA, extrachromosomal DNA, in many types of enterobacteria. In these experiments, CaCl ₂ - special cells in the bacterial population of the treatment was found to cause preferential transformed.

In the meantime, an experiment using a eukaryotic cell was carried out substantially independently of an experiment using a prokaryotic cell. 196
In 2 years (Szybalska, EH and Szybalski, W. PNAS 48:20
26 (1962)) is a transformed mammalian cell, but one that has been transformed at such a low frequency that it is not possible to distinguish the transformed cell from the cell that has just inherited the natural diachronic inheritance. Was reported. Again, though by prokaryotic cells, further reports of eukaryotic transformation appeared in the literature.

[0004]

However, in the transformation of prokaryotic cells, the frequency of transformation and the stability of the obtained transformation are high because the plasmid is unlikely to be incorporated into the chromosomal DNA. Low. As a result, the co-transformants lost the acquired trait after several generations. In addition, these bacterial experiments require the addition of a gene promoter to the transforming DNA to obtain expression.

Also, the reported results for transformed eukaryotic cells often could not be reproduced by others. In addition, the low frequency of transformation, lack of understanding of the molecular basis for genetic expression, and lack of molecular hybridisation probes account for the lack of progress in this area. Studies on the resulting transformed eukaryotic cells were essentially limited to viral genes (Graham, F.
L. et al., Cold Spring Harbor Symp.Quant. Biol. 39: 637.
~ 650 (1975) and McCutchen, JH and Pagano,
JS, Jounal National Cancer Institute, 41: 351 ~ 3
57 (1968)).

[0006]

Means for Solving the Problems The present invention encodes a foreign DNA (A) molecule and a dihydrofolate reductase that imparts methotrexate resistance to eukaryotic cells, as a means for solving the above conventional problems. To provide a eukaryotic cell co-transformed by one molecule formed by binding to a DNA (B) molecule, which is an amplifiable gene, which foreign DNA (A) is mainly selectable. It encodes protein substances not related to various phenotypes such as interferon protein, insulin, growth hormone, coagulation factor, viral antigen or antibody, and one enzyme.

[Explanation of terminology] Before explaining the present invention, it will be helpful for understanding the present invention to set forth definitions of some terms to be used thereafter. Transformation refers to a method for the genotyping of recipient cells mediated by the introduction of purified DNA. Transformation is typically detected by a stable heritable change in the phenotype of the recipient cell that results in an alteration in either the biochemical or morphological properties of the recipient cell. Cotransformation means a method for transforming a recipient cell with one or more different genes. Cotransformation is mediated by the introduction of DNA corresponding to either a chemically uncoupled gene or a chemically coupled gene, with simultaneous and sequential changes in the genotype of the recipient cell. Including both. A proteinaceous substance means any biopolymer formed from amino acids.
Genotype refers to the genetic makeup of an organism as distinguished from its physical appearance. Phenotype refers to the observable properties of an organism as created by the genotype as well as the environment. A selectable phenotype is a phenotype that confers resistance that confers on an organism the ability to survive under conditions that extinguish all organisms that do not possess the phenotype. Examples include drug resistance, or the ability to synthesize some molecules required for cell metabolism in a given growth medium. As used herein, a selectable phenotype also refers to a new phenotype, eg, by passing through or being secreted by a cell, and by a functional assay, immunological assay or biochemical assay. Includes identifiable phenotypes such as the manufacture of substances that can be detected as type. Interferon protein means the protein-like portion of glycoprotein interferon, ie the portion that remains after removal of the sugar moiety. It contains the protein portion of interferon derived from human leukocytes, fibroblasts, or lymphoid germ cells. Chromosomal DNA means DNA that is normally associated with histones in the form of chromosomes that reside in the nucleus of eukaryotic cells. Transcription means the formation of RNA chains by the genetic information contained in DNA. What is translation m-RN
It refers to the process by which genetic information in the A molecule dictates the order of specific amino acids during protein synthesis.

More recently, however, eukaryotic cells transformed with foreign DNA encoding a selectable phenotype, particularly in mammalian cells, have been reported (Wigler, M). Et al., Cell 11: 223-232 (197
7)). This work can be extended to allow eukaryotic cells to be cotransformed, expressing the interalia that obtains foreign DNA accumulated in the chromosomal DNA of the nucleus of the eukaryotic cell. Resulting. More unexpectedly, it has been discovered that such foreign DNA can be expressed by cotransformants, resulting in a functional protein. In addition to prokaryotes, foreign
DNA has been steadily expressed for hundreds of generations, and the result can be attributed to the accumulation of foreign DNA in chromosomal DNA.

[Biosynthesis of proteinaceous substance by eukaryotic gene in prokaryotic cell] The eukaryotic cell of the present invention comprises a proteinaceous substance,
In particular, it offers great advances in the inclusion of bacterial systems in the commercial preparation of eukaryotic proteins such as interferon proteins, antibodies and insulin. Such advantages include the ability to use invariant genes encoding precursors for such proteinaceous materials. Following cell synthesis, the precursors are processed or transformed in eukaryotic cells to produce the desired molecule of biologically effective form. This phenomenon is well known for insulin, which is first produced in eukaryotic cells as prepiroinsulin which is converted into active insulin by appropriate peptide cleavage in the cell. Since prokaryotic cells lack the essential cellular machinery to convert preproinsulin to insulin, insertion of an eukaryotic gene taken up into insulin into prokaryotic cells results in the production of preproinsulin rather than insulin. Ah In the case of insulin, a relatively small and well characterized protein, this difficulty can be overcome by chemical synthesis of the appropriate gene,
Such approaches are essentially limited by the level of understanding of the amino acid sequence of the desired protein. Thus, for interferon proteins, coagulation factors, antibodies and uncharacterized proteins whose precise amino acid linkages are not yet known, the prokaryotic system probably is not well documented.

[Biosynthesis of proteinaceous substances by eukaryotic genes in eukaryotic cells] In contrast, since the eukaryotic system has a processing mechanism required by eukaryotic cells, such a disorder does not occur. Thus, the present invention provides co-transformed eukaryotic cells that produce desired proteinaceous substances such as interferon proteins, insulin, antibodies and the like without requiring a detailed molecular understanding of the amino acid sequence.

In addition to the problem of precursors having extra amino acids that must be removed to produce active proteins, important biological materials are denatured by chemical addition after synthesis and cleavage. Let's do it. Thus, for example, the interferons produced in humans are glycoproteins that contain sugar molecules in addition to proteins. If produced in bacterial cells, interferon lacks the sugar molecule that is added when interferon is made in human cells. In addition, proteinaceous materials made in bacteria include endotoxins that could be inflamed if the proteinaceous material was given to a mammal without significant purification. In contrast, interferons made in eukaryotic cells would be free of endotoxin. The invention therefore also provides cotransformed eukaryotic cells which produce compounds which cannot be produced in bacterial cells, including non-proteinaceous substances such as glycoproteins and proteinaceous substances.

[0012]

The eukaryotic cell of the present invention encodes a DNA (B) molecule associated with an amplifiable gene for a dominant selectable phenotype not derived from the eukaryotic cell and a proteinaceous substance of interest. Co-transformed with a foreign DNA (A) molecule present.
The thus transformed eukaryotic cells are continuously enriched with methotrexate (hereinafter abbreviated as mtx), which is a reagent that enables survival or discrimination of eukaryotic cells in which the amplifiable gene is amplified. Cultivated in the presence of concentration. The DNA (A) molecule is preliminarily chemically bound to the DNA (B) molecule and then inserted into the eukaryotic cell, or the DNA (A) molecule is the DNA in the eukaryotic cell. The foreign DNA (A) molecule is amplified in association with the amplification of the amplifiable DNA (B) molecule by chemically binding to the (B) molecule and in the eukaryotic cell. As the DNA (A) to be inserted into the eukaryotic cell of the present invention, particularly selectable phenotypes such as interferon protein, insulin, growth hormone, coagulation factor, viral antigen, antibody and some enzymes DNA encoding unrelated proteinaceous material is suitable. The eukaryotic cells of the invention are capable of synthesizing quantities of the desired proteinaceous and other substances that cannot be obtained by conventional techniques.

[Detailed Description] According to the present invention, foreign DNA
(A) is a DNA (A) containing a genetically encoded selectable phenotype that is not expressed by the cell without being acquired by transformation, and is not chemically bound to the foreign Foreign DNA (A) can be inserted into any eukaryotic cell by cotransforming the cell with DNA (B). The co-transformation is performed in a suitable growth medium and in the presence of selected conditions such that the only cells that survive or are otherwise modified are those that have acquired a selectable phenotype (see Figure 1). .

Although the practices discussed below relate to cultured eukaryotic cells of mammalian line, such as human blood cells, mouse fibroblasts, Chinese hamster ovary cells, and mouse teratocarcinoma cells. It is clear that it is generally applicable to all eukaryotic cells such as cells from birds such as chickens, yeast and fungal cells, cells from plants including cereals and flowers. Therefore, although the eukaryotic cells of the present invention are ultimately most suitable as mammalian cells, it should be understood that the eukaryotic cells of the present invention are applicable to all eukaryotic cells. is there.

The eukaryotic cell of the present invention is particularly suitable when a foreign DNA containing a gene encoding a proteinaceous substance which is not linked to a selectable phenotype is inserted into the eukaryotic cell. It is useful. Since such proteinaceous substances are characterized by the fact that they are not associated with a selectable phenotype, cells containing the encoding DNA are therefore associated with the destruction of transformed cells and their inclusion. It cannot be identified except by measuring quantity.

As examples of proteinaceous substances, the genes that are inserted and expressed in eukaryotic cells using the cotransformation method include interferon protein, insulin, growth hormone, coagulation factors, viral antigens, enzymes and antibodies. Including.

In some cases the DNA (A) and DNA (B) need not be purified to obtain accumulation and expression, but the DN
It is often preferred that A be purified prior to use in cotransformed cells. Such purification limits the potential for false results due to the presence of impurities and increases the probability that co-transformed cells can be identified and stably cultured. Also, although not essential, it is sometimes desirable that DNA (A) and / or DNA (B) be obtained by endonuclease cleavage of chromosomal donor DNA, eg, restriction of endonuclease cleavage of eukaryotic chromosomal DNA. Is. In addition, it is desirable that DNA (A) and DNA (B) be treated with calcium phosphate prior to being used in cotransformed eukaryotic cells. The method for treating DNA in this way with calcium phosphate is more fully described below. Finally, foreign DNA (A) is proportional to DNA (B) encoding a selectable phenotype that constitutes an excess of DNA (A), eg in the range of about 1: 1 to about 100,000: 1. Is preferably present in an amount of.

In a preferred embodiment of the invention, the foreign DN
A (A) and / or foreign DNA (B) is contacted with bacterial plasmid or phage DNA prior to use in cotransformed eukaryotic cells. In a particularly promising embodiment, foreign DNA (A) and / or DNA (B) is contacted with phage DNA and then incorporated into phage particles prior to cotransformation.

Although any DNA (B) encoding a selectable phenotype will be useful in the transformed eukaryotic cells of the invention, the experimental details described are particularly derived from herpes simplex virus. To a thymidine kinase (abbreviated as tk hereinafter) and a gene for adenine phosphoribosyl transferase (abbreviated as apt). In addition, a gene encoding a selectable phenotype linked to drug resistance, for example, a DNA containing a mutant dihydrofolate reductase (hereinafter abbreviated as dhfr) gene that confers resistance to methotrexate on cells ( B) extends the applicability of the transformed eukaryotic cells.

According to a preferred embodiment, the cotransformation is DNA (A) which is not physically and chemically bound to DNA (B).
And the DNA (A) is stably integrated into the chromosomal DNA in the nucleus of the cotransformed eukaryotic cell.

The eukaryotic cells of the present invention are obtained in any suitable medium which is limited only in the ability of the eukaryotic cells co-transformed in the medium to survive and / or be distinguishable. It By way of example only, the applicable medium for mouse fibroblasts that have acquired the tk gene is the HAT described more fully below. Also, co-transformation is carried out in the presence of selected conditions capable of surviving and / or distinguishing these cells that have acquired a selectable phenotype. Such conditions would include the presence of nutrients, drugs, or other chemical antagonists, temperature, etc.

The eukaryotic cell of the present invention comprises a foreign DNA (A) encoding a desired substance that can be recovered from the cell using techniques well known in the art. In addition, the cells may be capable of transcribing DNA (A) to form proteins or other desired substances that can be recovered again using well known techniques. Finally, the cells can be grown in culture and then harvested of proteins and other desired substances.

Although the desired proteinaceous substance identified above is a natural substance, the cotransformed eukaryotic cell may be equally useful in the production of synthetic biopolymers into which synthetic genes are assembled. Thus, the eukaryotic cell of the present invention produces a novel protein that does not yet exist. In addition, the eukaryotic cells of the invention are present in very small amounts or in impure form as if they were presently present but their isolation and / or identification could not be specifically fulfilled. It enables the production of proteins to be carried out. Finally, the eukaryotic cells of the invention allow the production of partially proteinaceous substances such as glycoproteins and other products, the synthesis of which is genetically directed.

In order to increase the amount of gene product formed in the cell, an insertion of a transgene amplification is carried out in eukaryotic cells. Amplification of a gene that is amplifiable by an amplified DNA (A) molecule and to a dominant selectable phenotype not otherwise expressed by the cell to insert an amplification of the foreign DNA (A) molecule into the eukaryotic cell The cells are cotransformed with a chemically unbound amplified foreign DNA (B) molecule corresponding to. This co-transformation process is carried out in a suitable medium and in the presence of cells that acquire a dominant selectable phenotype and / or in the presence of mtx capable of distinguishing the cells. Desirably, a dominant gene (DNA
This is because such cells that have acquired the highest number of (B)) only survive and / or are identified.
In the presence of successively higher concentrations of x. These cells then also contain an amplification of DNA (A). This approach can be used, for example, in the mutant dh that confers resistance to mtx on cells.
It is particularly suitable for the amplified insertion of an amplifiable gene that confers drug resistance to cells, such as the fr gene. The eukaryotic cells in which DNA (A) has been amplified and transformed are then used to produce increased amounts of the gene product encoded by DNA (A).

Alternatively, the foreign gene may be amplified and ultimately expressed by the eukaryotic cell by transforming the eukaryotic cell with the DNA molecule, and each DNA molecule containing a foreign DNA (A ) Is chemically linked to foreign DNA (B) corresponding to an amplifiable gene for a dominant selectable phenotype not normally expressed by eukaryotic cells. The chemical bond between DNA (A) and DNA (B) is in particular the form of the bond formed as a result of enzymatic treatment with ligase. Hybrids formed in this way
Transformation with DNA molecules is then possible, in suitable growth media, and for example, in which the copy of the amplifiable gene is amplified sufficiently large to allow these eukaryotic cells to survive.
And / or in the presence of continuously increasing concentrations in the range of 1: 1 to 10,000: 1, based on the moles of mtk capable of distinguishing the cells. Using this approach, amplifiable genes are amplified for a dominant selectable phenotype not otherwise expressed by cells and eukaryotic cells would otherwise die or be incapable of discriminating between cells. It is able to survive and / or discriminate in the presence of elevated concentrations of mtx that can complement the resulting amplifiable gene. Although various amplifiable genes for a dominant selectable phenotype are useful in the eukaryotic cells of the invention, genes with drug resistance, such as the gene for dhfr, which confers resistance to mtx on cells, are particularly suitable. There is.

[0026]

By using co-transformed eukaryotic cells, amplification of proteinaceous or other desired molecules can be created in eukaryotic cells. For example, an interferon protein, insulin, growth hormone, coagulation factor, viral antigen, or antibody, or an amplification molecule of interferon itself can be produced in a eukaryotic cell cotransformed with hybrid DNA. Thus, or in the manner described below, the desired substance may be produced by eukaryotic cells, especially mammalian cells, which have been co-transformed with purified DNA treated with calcium phosphate. Thus, the eukaryotic cells of the present invention are capable of producing highly desired, rare and expensive proteinaceous and other biological materials at concentrations that would not be obtainable using conventional techniques. Is.

The eukaryotic cells of the present invention are also normally eukaryotic, such as glycoproteins, including interferons, which are partially proteinaceous, but which also contain other chemical species such as sugars, ribonucleic acids, histones and the like. It is possible to produce substances that are produced in very small amounts in cells. Although there is little understanding of how to produce biological substances whose intracellular synthesis is complicated by cytoplasmic substances such as glycoprotein, the use of the eukaryotic cells of the present invention allows commercialization. It is expected that it will be possible to synthesize such materials in commercially useful amounts. In particular, if one or more genes encoding the protein part of a cytoplasmic substance containing a non-protein part such as glycoprotein is inserted into a eukaryotic cell that normally produces such a substance, , The eukaryotic cell will not only produce the corresponding proteinaceous substance, but will also utilize existing cellular machinery to process the proteinaceous substance if the need expands, and it will also be suitable non-proteinaceous substances. Would be added to form the fully biologically active substance. Similarly, the eukaryotic cell can produce a biological substance containing a non-protein portion such as interferon and insulin in a structure having a biologically complete activity.

[0028]

EXAMPLES In the present invention, and as more fully described below, eukaryotic cells have been stably transformed with precisely defined prokaryotic and eukaryotic genes for which there are no selection criteria. Addition of the purified viral tk gene to mouse cells lacking this gene resulted in the appearance of stable transformed mouse cells that could be selected for their ability to grow in HAT medium. As a result. Since these biochemical transformations represent a subpopulation of cells with sufficient capacity that they will likely accumulate other unlinked genes at a higher frequency than the general population, cotransformation experiments will tk
Gene, bacteriophage ΦX174, plasmid
pBR322, or cloned chromosomes with human or rabbit β-globin gene linkage.

Tk transformation is further enhanced by contaminating hybrids
It is cloned and analyzed for DNA chain cotransformation. Mouse cell lines were identified as containing Φ X, pBR322, or human and rabbit β-globin linkage amplification. Chains cotransformed 1 to 50 times or more are high molecular weight DNs isolated from independent clones
Collected in A. Analysis of subordinate clones demonstrates that the cotransformed DNA is stable for many generations in culture. This cotransformation system makes it possible to introduce and stably integrate virtually any defined gene into cultured eukaryotic cells.
No ligation to either viral vectors or selectable biochemical markers is required either. Cotransformation with a dominant marker makes it possible in principle to introduce virtually any cloned genetic element into wild-type cultured eukaryotic cells.

At the end of this, dominant-acting, mtx-resistant, dhfr gene from CHO A29 cells was transformed into wild-type cultured mouse cells. CHO dh in transformants
Demonstrating the presence of fr linkage provided conclusive evidence for gene transformation. Exposure of these cells to elevated levels of mtx results in increased resistance to the drug, with amplification of the newly transformed gene. The mutant dhfr gene is therefore CHO A29 cell DN prior to transformation.
It has been used as a eukaryotic vector by ligating A into the pBR322 linkage. Amplification of the dhfr linkage results in amplification of the pBR322 linkage. The use of this gene as a dominant-working vector in eukaryotic cells would expand the repertoire of potentially transformable cells without limiting these families to mutants available for research anymore.

Using the procedure described, the cloned chromosomal rabbit β-globin gene has been introduced into mouse fibroblasts by DNA-mediated gene transformation. Co-transformed mouse fibroblasts containing this gene provide a unique opportunity to study expression in heterologous hosts and the post-processing of these linkages. Solution hybridisation experiments coordinated with RNA contamination (infection) techniques demonstrated that rabbit globin linkage was polyadenylated in cell lines transformed at least once.
It is shown to be expressed in cytoplasm as S species. These 9S chains result from perfect splicing and transfer of the two. However, these results indicate that fibroblasts from heterologous races contain the enzymes necessary for the precise processing of linkages between rabbit genes whose expression is restricted to Eristoblast. Imply that. Amazingly,
However, the 45 nucleotides present at the 5'end of the mature rabbit m-RNA are absent from the globin m-RNA linkage detected during the cytoplasm of the transformation assay. These studies show the potential value of the cotransformation system in the analysis of eukaryotic gene expression. Introducing a wild-type gene into cells cultured with native and in vitro assembled mutant gene provides one assay for the organoleptic significance of connective tissue.

Gene Amplification Recombinant DNA technology facilitates the isolation of some higher eukaryotic cells for which hybridisation probes can be used. For example, from 1 to 20 for these genes in cells that code for basal metabolic function
Genes that are expressed at abnormally low levels by mRNA transcription present as individual copies are simply isolated by conventional techniques involving the assembly of cDNA clones and the final screening of recombinant libraries. I can't. An improved approach to the isolation of such rarely expressed genes has therefore been developed to utilize transformation in concert with a procedure known as plasmid rescue. This overview of what is currently underway in the laboratory is outlined below.

The chicken aprt gene is an enzyme, Hin III or Xb.
Transforming aprt ^- mouse cells with cytoplasmic DNA that is not cleaved by a and is digested with these enzymes results in an aprt ⁺ clone expressing the chicken aprt gene. The chicken DNA cleaved with Hin III was replaced with Hin III
Ligation into the plasmid cleaved at pBR322 results in the formation of a hybrid DNA molecule in which the aprt gene is now flanked by plasmid chains. Transformation of arpt ^- cells is now done with this DNA. The transformation should contain the aprt gene covalently linked to pBR322 with this complete complex integrated in high molecular weight DNA in mouse cells. This first cell transformation serves to remove the chicken aprt gene from the vast majority of other chick linkages. This transformed cellular DNA is now pB
An enzyme that does not cleave either R322 or the aprt gene,
Treated with Xba 1 DNA. The resulting fragment is then circulated by ligation. One such fragment should include the aprot gene covalently linked to the pBR322 linkage encoding the prototype and ampicillin resistance markers.

Transforming bacteria such as Escherichia coli with these transfer markers now selects for plasmid linkage from eukaryotic DNA linked to chicken aprt linkage. This double-selection technique allows the isolation of genes that express at low levels in eukaryotic cells where hybrid hybridization probes are not readily available.

EXPERIMENTAL The results of various experiments are described herein to aid in better understanding of the eukaryotic cells of the present invention. The ability to transfer the purified gene into cultured cells provides a unique opportunity to study the functionality and physical status of foreign genes in transformed hosts.

[Transformation using tk gene as a marker]
Development of a system for transfer to HSV tk mutant mouse cells has been described (Wigler, M. et al., Cell 11: 223-232 (197
7)), allowing these studies to be extended to unique cellular genes (Wigler, M. et al., Cell 14: 725-731 (1
979)). High molecular weight DNA obtained from tk ⁺ tissue and cultured by cells from a variant of eukaryotic tissue can be used to transfer tk activity to mutant mouse cells lacking this enzyme. The generalization of the transformation process is the cellular aprt gene and hypoxanthine phosphoribosyl.
Demonstrated by the continuous transfer of the transferase (abbreviated as hprt hereinafter) gene (Wigler, M. et al., Proc.
Nat. Acad. Sci USA 76: 1373-1376 (1979); Willicke,
K. et al., Molec. Gen. Genet. 170: 179-185 (1979); Gra
Somatic Cell Genetics, f, LY, etc., printing (1979)
). More recently, it has been demonstrated that cells transformed with genes encoding selectable biochemical markers also frequently accumulate other non-physically linked DNA fragments. Here, the tk gene was co-transformed with defined prokaryotic and eukaryotic genes into cultured mammalian cells and used as a marker to identify the mammalian cells (Wigler, M. et al. , Cell 16: 777 ~
785 (1979)).

[Transformation with dhfr gene as marker]
The detection of gene conversion has in the past relied extensively on the availability of suitable mutant cell lines. In some cases, the mutant gene contains a dominant acting cell resistance to the metabolic inhibitor. Co-transformation with a dominant-acting marker makes it possible in principle to introduce any cloned genetic element into wild-type cultured cells. In this study, cells were transformed with a gene encoding a mutant dhfr gene that confers high levels of mtx on cell resistance (Flintof
f, WF, etc., Cell 2: 245-262 (1976)).

Cultured mammalian cells are very sensitive to the folate antagonist mtx. mtx-resistant cell lines have been identified and placed in three categories: (1)
Cells with reduced transport of this drug (Fischer, G.
A., Biochem. Pharmacol. 11: 1233-1237 (1962); Siro
Tuak, FM, etc. Cancer Res. 28: 75-80 (1968)):
(2) Cells with structural mutations that reduce the affinity of dhfr for mtx (Flintoff, WF et al., Cell 2: 245-
262 (1976)) and (3) cells that produce abnormally high levels of dhfr (Biedler, JL et al., Cancer Res. 32: 153-
161 (1972); Cha

Mtx resistant variant cell line A29 of interest is mtx
It was confirmed to synthesize elevated levels of the mutant dhfr with a reduced affinity for (Wigle
r, M. et al., Cell 16: 777-785 (1979)). Genomic DNA from this cell line was used in experiments as a donor to transfer the mutant dhfr gene to mtx-sensitive cells. Exposing mtx-resistant transformants to increased levels of mtx selects for cells that have amplified the transferred gene. Eukaryotic cells transferred in this manner allow for the conversion and amplification of virtually any genetic element in cultured mammalian cells.

[Mouse cell mutant hamster
Transfer of dhfr gene] High molecular weight cellular DNA was prepared from wild-type mtx-sensitive CHO cells and A29 cells, an mtx-resistant CHO derivative that synthesizes increased levels of mutant dhfr (Flintoff, WF et al., Cell 2: 245. ~ 262 (1976)). dh
tk ^- mouse L cells with either fr gene or tk gene
^{(Ltk - aprt -)..} .. The ability of these DNA preparations with respect to transition to were tested using a modified calcium phosphate co-precipitation method (Wigler, M. etc., Proc Nat Acad Sci USA
76: 1373-1376 (1979)). Mutants A29 and DNA from both wild-type CHO cells Ltk the tk gene ^- aprt ^-
It was effective in transferring to cells. The mtx resistant population was observed only by the following treatment of cells with DNA from A29. The data obtained show that treatment of mtx-sensitive cells with A29 results in the transfer and expression of the mutant dhfr gene.

To test this hypothesis directly, a molecular crossbreeding study was conducted to deduce from the putative transformations.
It was demonstrated that the hamster dhfr gene was present in the DNA. Mouse dhfr cDNA clone (pdfr-21) with homology to the structural genetic linkage of the hamster dhfr gene (Chan
g, ACY et al., Nature 275: 617-624 (1978)) was used to detect the presence of this gene in our transformation.

Limited analysis of the dhfr gene from A29, from putative transformations and from amplified mouse cells was performed by contaminating outcrosses (Southern, E.
M., J. Mol. Biol. 98: 503-517 (1975)). The DNA was cleaved by the restriction endonuclease HindIII, electrophoresed on an agarose gel and transferred to a nitrocellulose filter. These filters are then crossed by the high specific activity of ³² P-labelled nick-converted pdhfr-21 and developed by automated radiography: this method is a limited fragment of genomic DNA homologous to the dhfr probe. To visualize. Prominent band for mouse DNA
15 kb, 3.5 kb, and 3 kb, 7.9 for hamster DNA
Observed at kb, 3.7kb and 1.4kb. The limiting profiles between these two species differ greatly enough, making it possible to distinguish hamster genes in the presence of the endogenous mouse gene. The resistance of five L cell transformants to mtx is therefore tested by contaminating outcrosses.
In each transformed cell line, the endogenous mouse dh
An expected profile was observed with a band resulting from cleavage of the fr gene and a series of additional bands whose molecular weights were identical to those observed based on cleavage of hamster DNA. 17.9 kb observed in hamster DNA,
The 7.9 kb and 1.4 kb bands are diagnostic of the presence of the hamster dhfr gene and are present in all transformations.

In the first experiment, the lowest concentration of mtx (0.1
μg / ml) was chosen, which gives Ltk ^- aprt ^- cell survival of 10
^-7 or less. Previous research (Flintoff, WF, Ce
II 2: 245-262 (1976)) implied that the presence of a single mutant dhfr gene could confer cell resistance to this concentration of mtx. Comparison of the intensity of the hamster dhfr gene fragment of transformed cell DNA with that of wild-type hamster DNA suggests that our transformation contains at most one mtx-resistant hamster gene. By comparison, donor A29 cells (Flintoff, WF, et al. Cell Cell) were shown to produce elevated levels of the mutant dhfr.
2: 245-262 (1976)) appear to contain amplification of this gene.

Amplification of the transferred dhfr gene The first transformation was selected for resistance to relatively low levels of mtx (0.1 μg / ml). For all clones, however, it is possible to select for cell resistance to elevated levels of mtx by exposing the mass medium to successively increasing concentrations of this drug . In this method, we first use 0.1 μg / ml
Starting from clones that were resistant to mtx
Culture-resistant strains up to 40 μg / ml were isolated. We then asked whether the increased resistance to mtx in these transformants was associated with amplification of the dhfr gene, and if so, whether the endogenous mouse or newly transferred hamster genes were amplified. I checked. DNA from four independent isolates and their resistant derivatives were tested by contaminating outcrosses. In each case, increased resistance to mtx was accompanied by an increase in hamster gene copy number. This is 1.
It is most easily seen by comparing the intensities of the 5 kb band. In each case, we have an endogenous mouse
No amplification of the dhfr gene was detected. Finally, it should be noted that not all lines selected at mtx equivalent concentration have the same dhfr gene copy number.

“Dhfr Gene as a Generalized Transformation Vector” The selectable gene can be used as a vector to introduce other genetic elements into cultured cells. Previous studies have demonstrated that cells transformed with the tk gene are likely to combine with other non-chemically linked genes (Wigler, M. et al. Cell 1
6: 777-785 (1979)). A generalization of this approach was tested against the selectable marker, mutant dhfr gene. 20 μg of total cellular DNA from A29 was mixed with 1 μg of HindIII linearized pBR322 DNA. Recipient cells were exposed to this DNA mixture and, after two weeks, mt
x Resistant population was removed. Genome DN from transformants
A was isolated, cleaved with HindIII and analyzed for the presence of pBR322 linkage. Two independent isolates were tested in this way, and in both cases pBR3
A 22-link amplification was present in these mtx transformants.

An improved approach to generalized transformation involves ligation of nonselectable DNA chains. Since the mutant dhfr gene is dominant in acting on drug resistance factors, this gene is an ideal vector.
Furthermore, it is possible to amplify any genetic element ligated to this vector by selecting for cell resistance to elevated levels of mtx. To test this possibility, restriction endonucleases that do not disrupt the A29 dhfr gene were identified by transformation assays. One such restriction endonuclease, Sal I, is A29
Does not destroy the transforming power of DNA. Cleaved with Sal I
A29 DNA was therefore ligated with an equal amount of Sal I linearized pBR322. This ligation product was then used in transformation experiments. The mtx resistant population was isolated and 0.1
Grown in trout medium at μg mtx / ml. The trout culture was then exposed to increasing concentrations of mtx. DNA is 0.
Obtained from mass culture resistant to 1, 2, 10 and 40 μg / ml mtx, pBR322 and dhfr linkage copy numbers were determined by contaminating intercrosses. Six independent transformed lines were tested in this way. Five of these lines displayed amplified bands homologous to the pBR322 linkage. In four of these transformed clones, at least one specific band of pBR322 increased in intensity in the amplification of dhfr. In SS-1, the two specific bands of pBR322 were 0.1
It was observed in DNA from cell resistant to μg / ml mtx. These bands increase several-fold in intensity in cell resistance to 2 μg / ml. However, 40 μg /
No increase in strength was observed in cells selected for resistance to ml. 0.1 for the second line
The SS-6, all pBR322 bands present at μg / ml continue to increase in intensity as cells were first selected at 2 μg / ml of mtx and then at 40 μg / ml. Curiously, a particular band of new pBR322 appears after selection at high mtx concentrations. It was assessed that there was at least a 50-fold increase in copy number for pBR322 linkage in this cell line. In the third cell line, HH-1, two specific bands of pBR322 increase in intensity upon amplification and the others remain constant or decrease in intensity. Thus, the pattern of amplification of pBR322 linkage observed in these cells can be completely altered. However, the mutant dhfr gene can be used as a vector to introduce and amplify limited DNA linkages into cultured animal cells.

[Discussion] DN in the study of eukaryotic gene expression
The potential utility of A-mediated transformation depends on its large extent due to its generalization. Cellular genes encoding selectable biochemical functionalities have been introduced into previously mutated cultured cells (Wigler,
M., et al., Cell 14: 725 ~ 731 (1979); Wigler, M., et al.,
Proc. Nat. Acad. Sci. USA 76: 1373 ~ 1376 (1979); W
illecke, K., et al., Molec. Gen. Genet. 170: 179-185 (1
979); Graf. LH, et al., Somatic Cell. Genetics, printing (1979)). In a recent study, a dominant-acting mtx-resistant dhfr gene was transferred to wild-type cultured cells.
The use of this gene as a vector in a cotransformation system will now allow virtually any genetic element to be introduced into the host in a new cellular environment.

In the first experiment, DNA from A29 cells, an mtx-resistant CHO derivative that synthesizes the mutant dhfr, was transformed into mouse L
Added to the culture of cells. 1 for mtx resistant population
From 10 populations / 5 × 10 ⁵ cells / 20 μg cell DNA. This DNA was a potent donor of the tk gene, although no population was observed upon transformation with DNA obtained from wild-type mtx-sensitive cells. The definitive evidence that we resulted in the transfer of the mutant hamster dhfr gene was obtained by demonstrating the presence of the hamster gene in mouse transformants. The limited maps of the mouse and hamster dhfr genes differ markedly and make it possible to distinguish these genes in contaminated outcross experiments. In all the transformations tested, two sets of restriction fragments homologous to the mouse dhfr cDNA clone, a series of bands characteristic of the endogenous mouse gene,
And a second series of bands characteristic of the donor hamster gene is observed.

The utility of dhfr locus transformation is a function of the relative frequency of both transformation and natural resistance to mtx. Demonstration that all isolated mtx-resistant L cells result from transformation rather than amplification of endogenous genes suggests that amplification of dhfr is a rare event in this cell line. Attempts have been made to transform other cell lines, including mouse teratoma and rat liver cells, and in this example cross-bred studies revealed that acquisition of mtx resistance resulted from amplification of the endogenous dhfr gene. Become. Purified dh
The use of the fr gene is probably to overcome these difficulties by significantly increasing the frequency of transformation.

Dh observed in the first transformant
The copy number of fr is low. This observation suggests that a single mutant dhfr gene can confer cellular mtx resistance under selected criteria (0.1 μg / ml mtx) in previous studies (Fl
intoff, WF, etc., Cell 2: 245-262 (1976)). Exposure of these initial resistant transformants to escalating drug concentrations resulted in selection of cells by increased mtx resistance resulting from amplification of the newly transposed mutant hamster dhfr gene. Wake up.
No amplification of the endogenous mouse gene was observed in any of the transformants depending on the pressure selected. A single mutant gene probably confers significantly greater resistance to a given concentration of mtx than a single wild-type gene. If the frequency of amplification is low, then one will select a resistant variant that has the fewest number of events of simply amplification. The newly transferred gene will be more easily amplified than the endogenous gene.

The mutant dhfr gene was used as a dominant transfer vector to introduce non-selectable genetic elements into cultured cells. One experimental approach was the previous observation that competent cells frequently accumulate other, physically uncoupled genes (Wigler, M.
Et al., Cell 16: 777-785 (1979)). pBR322DN
Cultures exposed to A cause an mtx-resistant cell line containing a complex copy of the bacterial plasmid with heritable DNA containing the mutant dhfr gene.

A modified approach to genetic vectoring allows selection of pBR322 linkages prior to transformation
Including ligation to the dhfr gene. This product is also complex
Results in a transformation containing the pBR322 linkage. dhfr amplification is pBR3
The result is 22-linked amplification, but the pattern of amplification varies by cell line. In one example, all pBR322 linkages amplify with increasing mtx concentration.
In other strains, a subset of linkages is amplified. In other lines, linkage appears to have been lost or rearranged. Amplification up to 40 μg / ml in some strains
It progresses with increasing mtx concentration, while in others the amplification is stopped at 2 μg / ml. Currently, the amplification process is not understood and the amplification units are not defined. It is clear that any mechanism responsible for these complex events can be developed to substantially regulate the administration of any gene that leads them into cultured cells.

[Materials and Methods] Materials and methods for carrying out the experiment are as follows. Cell culture Derivatives of Ltk clone D (Ltk aprt, Kit, S. et al., Es
p. Cell Res. 31: 291 ~ 312 (1963)) is 10% calf serum (Flow Laboratories, Rockville, MD).
And in Zlubeco's modified Eagle's medium (DME) containing 50 μg / ml diaminobulin (DAP). Prior to transformation, cells were washed and in the absence of DAP 3
It has been grown for generations. Transformed dhfr (mt
Chinese hamster cell line (Flintoff, WF, et al., Somati) containing A29mtx ^Riii (conferred to x)
c Cell Genetics 2: 245-261 (1976)) were propagated in DME supplemented with 3X-nonselective amino acids, 10% calf serum, and 1 μg / ml amitobuterin. For amplification experiments, the medium was supplemented with an additional 20 μg / ml methotrexate. Rodent Ltk ^- aprt cells are Ltk ^- clone D cells
It is an aprt negative derivative. Cells are nourished in growth medium and Wi
Prepared for transformation as described by gler, M., et al., PNAS 76: 1373-1376 (1979). Hep-2 (human),
HeLa (human), CHO (Chinese hamster ovary), and Ltk ^- cells were grown in growth medium.
LH2b, transformed with herpes simplex virus tkDNA
Ltk ^- derivatives hypoxanthine at 15μg / ml, 0.2 μg / m
Growth medium (HAT) containing aminobuterin at 1 and thymidine at 5.0 μg / ml (Wigler, M., et al. Cell 1: 223-23
2 (1977)). All medium dishes are Nunclon (Vanguard International, Neptune,
NJ) It was plastic. Donor-independent mouse tetratocalcinoma cell culture line 6050P (Watanabe, T., et al., PNAS 7 obtained from tumor of OTT 6050 transplant line)
5: 5113-5117 (1978)) was used as the wild type, or tk ⁺ parent, and is herein named TCC wt. This strain has an X / O sex chromosome type, a 39-chromosome pattern number with the characteristics described in Watanabe, T., et al. (1978). The cells were grown in Zulbecco's modified Eagle's medium containing 10% fetal calf serum. After 3 hours of exposure to 3 μg / ml mutagenizing N-methyl-N′-nitro-N-nitrosoguanidine, cells were allowed to recover for 2 weeks and then transferred to medium containing 80 μg / ml BrdUrd, a series of resistant clones were isolated, clonal lines used in this transformation experiments (TCC tk ^-) is supplied. This line has a biennial inheritance frequency to wild type of 10 ^-8 or less. Prior to transformation, cells should be BrdUrd 30 μg / ml
They were nourished in culture medium, washed and grown for three generations in the absence of the drug. Tk of ^- transformation efficiency mouse L over cells (Ltk) ^- It (Kit lack lineage, S, etc., Exp Cell.Res 331:... 297~312
(1963)).

[Extraction of Genomic DNA and Cleavage of Restriction Endonuclease] High molecular weight DNA was used for culturing cells (CHO, LH2
b, and HeLa) or as mentioned earlier (Wigle
r, M., et al., Cell 14: 725-731 (1978)), obtained from frozen rabbit liver. High molecular weight salmon sperm DNA was obtained from Worthington. Restriction endonuclease cleavage
(BamI, HindIII, KpnI, and XbaI) is 50 mM Nac
l, 10 mM Tris-Hcl, 5 mM MgCl ₂ , 7 mM mercaptoethanol, and bovine serum albumin at 100 μg / ml in buffer (pH 7.9). The enzyme to DNA ratio is at least 2 units / μg of DNA and the reaction mixture is 37
Aged at 0 ° C. for at least 2 hours. To monitor the completion of the digestion, 1 μl of nick-transformed adenovirus 2 [ ³² P] DNA was aged in a reaction volume of 5 μl for at least 2 hours and the cleavage product electrophoresed in a 1% agarose gel. , And digestion was monitored by exposing the dried gel to Kronex 2DC X-ray film. As described above, intact herpes simplex virus (HSV) DNA was isolated from CV-1-infected cells (Pellic
er, A., et al., Cell 14: 133-141 (1978)). 6 mM DNA
KpnI (New England, in a buffer containing Tris (pH 7.9), 6 mM MgCl ₂ , 6 mM 2-mercaptoethanol, 6 mM NaCl and 200 μg / ml bovine serum albumin.
Biolab). Limited DNA is 0.
24 hours via 5% agarose gel (17 x 20 x 0.5 cm)
Fractionated by electrophoresis 70V, and 5.1kb of tk ^-
Fragments containing Maxam, AM and Gilbert, M. PNAS 74: 5
60-564 (1977), and Wigler, M., et al., Cell 14: 725.
~ 731 (1978)) and extracted from the gel. The φX 174am3R FIDNA was purchased from Bethesda Research Laboratories. Plasmid pBR32
2 DNA was grown in E. Coli HB 101, Clewell, D.
B., J. Bacteriol. 110: 667-676 (1972). The cloned rabbit β giant globin gene in the λ Sharon 4A derivative (RβG) was identified and isolated as described previously (Maniatis, T.,
Et al., Cell 15: 687-701 (1978)). In the amplification experiment,
The size of the high molecular weight DNA is 0.3 using herpes simplex virus DNA and its XbaI fragment as a marker.
Measured by electrophoresis in a% agarose gel. Only DNA with an average size greater than 75 kb was found to be transforming in amplification experiments. In this experiment, plasmid DNA was isolated from chloramphenicol-amplified cultures by isobaric centrifugation in a CsCl gradient containing 300 μg / ml ethidium bromide.

[Transformation and selection] The transformation record is Gr
aham, FL and Vander Eb, AJ, Virology, 52: 4
56-457 (1973) with the following modifications. One day before transformation, cells were 0.7 per dish.
Seed at × 10 ⁶ cells. The medium was changed 4 hours before transformation. Sterile, ethanol-precipitated high molecular weight or restriction endonuclease-cleaved eukaryotic DNA lysed in 1 mM Tris (pH 7.9) /0.1 mM EDTA
DNA in [mu] g / ml, and a 250mM CaCl ₂ DNA / CaCl ₂
It was used to adjust (Marine croto). A two-fold concentrated Hepes buffered salt (2XHBS) was prepared. It was adjusted to pH 7.10 ± 0.05 with 280 mM NaCl, 50 mM Hepes, and 1.5 mM sodium phosphate. DNA / C
The aCl ₂ solution was added dropwise to the same volume of sterile 2X HBS. A 1-ml sterile plastic pipette with a cotton plug was inserted into a mixing tube containing 2X HBS and air bubbles were introduced by bubbling while DNA was added. The calcium phosphate / DNA precipitate is allowed to form at room temperature for 30-45 minutes without stirring. The precipitate was then mixed by gently picking it up in a plastic pipette and 1 ml of precipitate per dish was added directly to 10 ml of growth medium covering the recipient cells. After aging at 37 ° C for 4 hours, the medium is replaced and the cells are aged for an additional 20 hours. The selective pressure is then exerted. For tk ⁺ selection, the medium is changed to growth medium containing HAT. For aprt ⁺ selection, cells were trypsinized and at low density (approximately 0.
5 × 10 ⁶ cells) were replaced with a medium containing 0.05 mM azaserine and 0.1 mM adenine. For both tk ⁺ and aprt ⁺ selection, the selective medium was changed the following day, after 2 days, and then every 3 days for a few weeks during which the transformant clones were occurring. Populations were excised using cloning cylinders, and the rest of the population was recorded after formaldehyde fixation and staining with Diemsa. For characterization, clones were subsequently grown in mass medium under selective pressure. Records were kept for the apparent number of cells doubled for each clone isolated.

Mtx-resistant transformants of Ltk ^- aprt ⁺ cells are A
Transformation with high molecular weight DNA from 29 mtx ^Riii cells and
Obtained after selection in DME containing 10% fetal calf serum and 0.2 μg / ml amethopterin. For tk ⁺ selection, cells were grown in HAT medium due to resistance to methotrexate and cells were selected in medium supplemented with 0.1 μg / ml methotrexate. Each population was cloned from an individual dish to ensure that each transformant resulted from an independent event. A29 DNA and linearized pBR322 DNA
The ligature with Sal under the conditions recommended by the supplier.
It was prepared by aging a 1: 1 ratio ( ^W / _W ) of I-cleaved DNA to T ₄ ligase (Bethesda Research Laboratories). Calcium phosphate precipitation is 2μ
Prepared with g ligature and 18 μg carrier / ml and added to recipient cells (the amount of ligation is limited due to the observation that the plasmid inhibits transformation). The DNA was kept in contact with the cells for 4-12 hours, and the medium was then aspirated and replaced with fresh DME. Selective pressure was applied for 24 hours and then exposed to DNA. After 2-3 weeks the population was isolated using cloning cylinders.

[0057] In mice tetra solved Resid Norma cell experiments, transformation TCC tk ^- cells 3 × 1 day prior to transformation
The transformation was performed as described above except that the cells were seeded at 10 ⁵ cells / dish. For each dish of adherent cells, 4 μg of calcium phosphate / DNA precipitate conditioned with recombinant plasmid, digested with Bam H1 in the presence of 20 μg of high molecular weight DNA obtained from Ltk ^- aprt ^- cells. Ptk-1
Was added. In addition, some cells were treated with Sunpension (Willecke, K., et al., Molec. Genet.
170: 179 ~185 (1979)) 7 × 10 6 freshly trypsinized been TCC tk ^- plasmids Pt cells that are Bam H1 over cleaved
10 μg DNA from k-1 and high molecular weight DNA from salmon sperm
Of 150 μg of calcium phosphate / DNA precipitate prepared. After centrifugation, resuspension, and shaking, cells were re-thinned in growth medium as described in Willecke, K., et al. (1977). After 3 days, the medium was replaced with HAT medium and the transformant population was isolated after 2 weeks.

Cotransformation experiments were performed on the HindIII-cleaved plasmid pHβ-8 containing the chromosomal adult β-globin gene.
Performed with 4 μg of Bam H1-digested Ptk-1 DNA together with 4 μg (Lawn, RM, et al., Cell 15: 1157-1174 (197).
8)). Tk ⁺ transformant is 0.1 mM hypoxanthine / o.4 μM
Selected in growth medium containing aminobuterin / 16 μM thymidine (HAT). Populations were excised with cloning cylinders and grown in trout medium.

[Cotransformation of HSVtk gene with defined DNA linkage] Ltk ^- aprt ^- mouse cells were expressed as 1-10 μg of Φ17.
As described above in the presence of 1 μg of HSV-1 gene and 10-20 μg of salmon sperm carrier DNA with either 4,1 μg pBR322 or 1 μg of RβG-1 DNA (Wigler,
M., et al., PNAS 76: 1373-1376 (1979)).
Tk ⁺ transformation was selected in DME containing hypoxanthine, aminobuterin, and thymidine (HAT) and 10% calf serum. Isolated populations were excised using cloning cylinders and grown in mass medium.

[Enzyme Assay] Extract is 0.5% Triton X
It was obtained by resuspending washed cell granules (about 10 ⁷ cells) in 0.1 ml of 0.02 M potassium phosphate, pH 7, containing -100. The supernatant (cytoplasm) obtained 25 minutes after 700 × g centrifugation was used for quantification of enzyme activity and electrophoresis. Aprts and proteins were assayed as previously described (Chasin, LA, Cell 2: 37-41 (1974)).
Inhibitor of 3 mM thymidine triphosphate, 5'-nucleotidase in the reaction mixture (Murray, AW
And Friedrichs, B., Biochem, J. 111: 83-89 (196
The inclusion of 9)) did not increase AMP recovery, which indicates that nucleotidase does not interfere with the measurement of aprt activity. The conductivity focusing of aprt was essentially described for hprt (Chasin, LA and Urlaub, G. Somat. Cell Gene
t. 2: 453-467 (1976)) with the following exceptions. Amphorin (LKB) 0.8% of mixed solution pH2.5〜4, 0.8%
To assay the enzyme activity of polyacrylic amide gel containing pH 4-6, and 0.4% pH 5-7, [2- ³ H] adenine was used.
[0.04mM, 1Ci / mmd, New England Nuclear (1Ci = 3.7 × 10
¹⁰ Beklem)] was substituted for hypoxanthine.

[Th activity assay] To measure specific activity,
Cells from monolayer cultures were scraped into phosphate buffered serum and washed. Cell granules have 5 volumes of extraction buffer (0.01M Tris-HCl, pH7.5, 0.01M KCl, 1mM MgCl ₂ ,
1 mM 2 mercaptoethanol and 50 μM thymidine). The cell suspension was frozen and thawed 3 times and the KCl concentration was then adjusted to 0.15M. After sonication, the cytoplasm extract is 3
Obtained by centrifugation at 0,000 Xg for 30 minutes, and the supernatant was used for the tk assay as described in Wigler, M. et al., Cell 16: 777-785 (1979). Cytoplasmic extracts from tumors were obtained after cell disruption in a Potter-Elve Jame homogenizer. They were then treated as described above for cells cultured. One unit of tk is defined as the amount of enzyme that converts 1 nmole of thymidine to thymidine monophosphate per minute. In the enzyme nulling study, anti-HSV-1tk anti-serum or preimmune serum was mixed with an equal volume of cytoplasm extract and ATP and magnesium were added to 6.7 mM. The enzyme-antibody mixture was aged at room temperature for 30 minutes, 2000X
Centrifuged for 10 minutes, and the supernatant was assayed for tk activity. In a further biochemical assay, 30,000 Xg supernatants of homologues from cell culture and from solid tumors were obtained.
Electrophoresed on a 1.6 mm thin 5% polyacryl amide gel and cut by Lee, LS and Cheng, Y.
Assayed for tk activity as described in C., J. Biol. Chem., 251: 2600-2604 (1976).

[RNA isolation] Total RNA was treated with phenol at pH 5.1, phenol / chloroform / isoamyl alcohol (25: 24: 1, vol / vol), and chloroform / isoamyl alcohol (24: 1, vol / vol). Isolated from logarithmic phase cultures of L cells transformed by successive extractions. After ethanol precipitation, the RNA
Digested with DNase (Maxwell, IH, etc., Nucleic
Acids Res. 4: 241-246 (1977)) and ethanol precipitated. Nuclear and cytoplasmic blasts are Wigler,
M., et al., PNAS 76: 1373-1376 (1979), was isolated as described and RNA was extracted as described above. Cytoplasm polyadenylated RNA was isolated by oligo (dT) -cellulose chromatography (Axel, R., et al., Cell 7: 247-254 (1976)).

[CDNA synthesis] Rabbit and mouse cDNA
By Myers, JC and Spiegelman, S., PNAS 75: 5329
~ 5333 (1978) as described in Avian Myeloblastosis Virus Reverse Transcriptase (R
NA-dependent DNA polymerase).

[Isolation of Transformed Cell DNA] Cells are exfoliated and harvested in PBS and centrifuged at 1000 × g for 10 minutes. Particles are TNE [10mM Tris-HCl (pH8.0), 150mM NaCl, 1
Resuspended in 40 volumes of 0 mM EDTA] and added SDS and proteinase K at 0.2% and 100 μg / ml, respectively. The lysate is aged at 37 ° C. for 5-10 hours and then extracted sequentially with buffer saturated phenol and CHCl ₃ . High molecular weight DNA was isolated by mixing the aqueous phase with 2 volumes of cold ethanol and immediately removing the formed precipitate. The DNA was washed with 70% ethanol and dissolved in 1 mM Tris, 0.1 EDTA. The cytoplasm from clones ΦX4 and ΦX5 is Rigold, G.
M., et al., Cell 10: 19-26 (1977). The fraction is further Hirt, B., J. Mol.
As described in Biol. 25: 365-369 (1967), it was fractionated into high-molecular weight and low-molecular weight DNA.

DNA Filter Interbreeding Cellular DNA was treated with restriction endonucleases, electrophoresed on agarose slab gels, transferred onto nitrocellulose filter sheets, and Wigler, M., et al., PNAS 7
³² P-labeled DNA as described in 6: 1373-1376.
Intercrossed with the probe. DNA from transformed cells
Was digested with various restriction endonucleases using conditions specified by the supplier (New England Biolab or Bethesda Research Laboratories). Degradation at an enzyme-to-DNA ratio of 1.5 U / μg
It was performed at 37 ℃ for 2 hours. The reaction was terminated by the addition of EDTA, and the product was 36 mM Tris, 30 mM NaHPO ₄ , 1 mM E.
Electrophoresed on a horizontal agarose slab gel in DTA (pH 7.7). The DNA fragments were transferred to nitrocellulose sheets, cross-bred and washed by the method previously described and two modified methods (Weinstock, R., et al., PN.
AS 75: 1299 to 1303 (1978)). Two nitrocellulose
Filters were used during the transition (Jeffreys, AJ and Flavell, RA, Cell 12: 1097-1108 (1977)). The lower filter was discarded and then crossbred, the filter was 2XSSC, 25 mM sodium phosphate, 1.5 mM Na ₄ P ₂ O ₇ , 0.05%.
Washed 4 times for 20 minutes in SDS, and then 1:
Sequential washes in this buffer diluted 1 and 1: 5 (Jeffreys, AJ and Flavell, R.
A., Cell 12: 429-439 (1977)). In the amplification experiment,
The probe is pBR322 or pdhfr- with ³² P-nick conversion.
21, any of the cDNA copies of mouse dhfr m-RNA (Chang, ACY, et al., Nature 275: 617-624 (197
8)).

[Solution cross-breeding] ³² P-labeled globin cDNA (2.9 × 10 ⁸ cpm / μg specific activity) was 0.4 M NaC.
l / 25mM 1.4-Piperazine Jetane Sulfonic Acid (P
ipes), pH 6.5 / 5mM EDTA, at 75 ° C crossed with excess RNA. Aging time does not exceed 70 hours. Rots
RNA nucleotide moles / liter / hour. Time is seconds,
Was calculated as Resistance to the single filamentous nuclease S1 was conferred in the outcross. The fraction of cDNA was measured as described (Axell, R. et al.,
Cell 7: 247-254 (1976)).

[RNA filter interbreeding] RNA (Baile
y, J. and Dabidson, N., Anal. Biochem 70: 75-8
5 (1976) ) and a 1% agarose gel (17 × 20) containing 5 mM methylmercury hydroxide.
X 0.4 cm). RN in each groove
The concentration of A was 0.5 μg / μl. Electrophoresis at room temperature 11
It was 0V for 12 hours. Alwine., JC, PNAS 74: 5350
~ 5354 (1979), RNA was prepared by diazotized cellulose using pH 4.0 citrate transfer buffer.
Transferred from the gel to paper. After transfer, the RNA filter is washed with transfer buffer containing carrier RNA at 500 μg / ml.
Aged for hours. The RNA on the filter was cross-crossed with 50 μg / ml of a cloning DNA probe labeled with a specific activity of 2-8 × 10 ⁸ cpm / μg by ³² P-nicko conversion (Weinstock, R., et al., PNAS 75: 1299-1303). (197
8)), the reaction volume was 25 μl / cm ² of the filter. Outcrosses were at 57 ℃ for 36-46 hours in 4X standard serum citrate (0.15M Nacl / 0.015M sodium citrate) / 50% formamide. After crossbreeding, the filters were two 2X standard serum citrate / 25 mM sodium phosphate / 1.5 mM sodium pyrophosphate.
/0.1% sodium dodecyl sulfonate / 5mM EDTA at 37 ℃ in 30 mM
Formamide was removed by dipping while shaking for minutes. Sequential washes were performed with 0.1X standard serum citrate containing 1X and 5mM EDTA and 0.1% sodium dodecyl sulfonate at 68 ° C, 30 each.
It was a minute.

[Birk Sharp Analysis of Rabbit β-Globin in Transformed Mouse L Cells] Crossbreeding was 80% (vol / vol) formamide (Eastman) /0.4MPip
es, pH 6.5 / 0.1 mM EDTA / 0.4 M Nacl (Casey, J. and Davidson, N., Nucleic AcidRes, 4: 1539-1552 (197
7)); (Berk, AJ and Sharp, P. A, Cell 12:72.
1-732 (1977)), 18 hours, 51 ° C. for 1.8 Kbp Hha I fragment and 49 ° C. for Psi 1 fragment. The outcrosses were treated with S1 nuclease and analyzed essentially by the method described in (Berk, AJ and Sharp, PA (1977)).

Although this disclosure describes all essential informational material relating to the present invention, many of the publications cited herein are helpful in understanding the context of the invention and aspects of the technology. Will. Accordingly, all cited publications are incorporated herein by reference in the present disclosure.

[Brief description of drawings]

FIG. 1 is a schematic process diagram illustrating a cotransformation process in the present invention.

FIG. 2 is a schematic process diagram illustrating a method for recovering foreign DNA from co-transformed and cultured cells using the double selection technique.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 38/27 38/43 38/21 39/00 A 39/395 A 45/02 B 8415-4C C12N 15/09 C12P 21/00 F 9282-4B E 9282-4B H 9282-4B C 9282-4B (C12P 21/00 C12R 1:91) A61K 37/26 37/36 37/465 37/48 37/66 B 9050-4B C12N 15/00 A (71) Applicant 593220742 Michael H. Wiggler MICHAEL H. WIGLER USA 11724 New York, Cold Spring Harbor Pea Box 100 Cold Spring Harbor Laboratories (71) Applicant 593220753 Sole Jay. Silbastine SAUL J. SILVERSTEIN United States 10533 New York, Irvine, Birch Lane 260 (72) Inventor Richard Axel United States 10025 New York, New York, Riverside Drive 410 (72) Inventor Michael H.C. Wiggler United States 11724 New York, Cold Spring Harbor PO Box 100 Cold Spring Harbor Inside Laboratory (72) Inventor Saul Jay. Sylvastine United States 10533 New York, Irving, Birch Lane 260

Claims (22)

[Claims] 1. A DN, which is an amplifiable gene encoding a foreign DNA (A) molecule and a dihydrofolate reductase that confers methotrexate resistance to eukaryotic cells.
Eukaryotic cells cotransformed with the A (B) molecule 2. The eukaryotic cell according to claim 1, wherein the foreign DNA (A) is inserted into the eukaryotic cell in a state of being preliminarily bound to the foreign DNA (B). 3. The eukaryotic cell according to claim 1, wherein the foreign DNA (A) and the foreign DNA (B) are separately inserted into the eukaryotic cell. 4. The eukaryotic cell according to claim 1, wherein the foreign DNA (A) encodes a protein substance that is not associated with a selectable phenotype. 5. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) encodes an interferon protein. 6. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) is a DNA encoding insulin. 7. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) is a DNA encoding growth hormone. 8. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) is a DNA encoding a coagulation factor. 9. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) is a DNA encoding a viral antigen or antibody. 10. The eukaryotic cell according to claim 4, wherein the foreign DNA (A) is a DNA encoding one enzyme. 11. The eukaryotic cell according to claim 1, wherein the foreign DNA (A) is substantially purified DNA. 12. The eukaryotic cell according to claim 1, wherein the foreign DNA (A) is DNA obtained by restriction endonuclease degradation of eukaryotic chromosomal DNA. 13. The foreign DNA (A) and / or the DNA (B)
The eukaryotic cell according to claim 1, which is bound to bacterial or phage DNA. 14. The foreign DNA (A) and / or the DNA (B)
The eukaryotic cell according to claim 1, wherein the eukaryotic cell is bound to the phage DNA encapsulated in the phage particle. 15. The foreign DNA (A) and / or the DNA (B)
The eukaryotic cell according to claim 1, wherein the cell is treated with calcium phosphate. 16. The eukaryotic cell according to claim 1, wherein the eukaryotic cell is a mammalian cell. 17. The eukaryotic cell according to claim 16, wherein the mammalian cell is erythroblast. 18. The eukaryotic cell according to claim 16, wherein the mammalian cell is fibroblast. 19. The foreign DNA (A) is 1: 1 with respect to the DNA (B) encoding a proteinaceous substance associated with a selectable phenotype.
To about 100,000: 1.
Eukaryotic cell described in 20. The eukaryotic cell according to claim 1, wherein the foreign DNA (B) encodes a proteinaceous substance associated with a selectable phenotype consisting of a gene associated with resistance to a drug or a chemical antagonist. 21. The eukaryotic cell according to claim 20, wherein the gene associated with resistance to a drug or a chemical antagonist is a gene encoding a mutant dihydrofolate reductase that changes cells to methotrexate resistance. 22. The foreign DNA (A) is a chromosome DN of the eukaryotic cell.
The eukaryotic cell according to claim 1, which is incorporated into A.