EP0116631A1 - Verwendung eukaryotischer promotorsequenzen bei der herstellung von proteinmaterialien - Google Patents

Verwendung eukaryotischer promotorsequenzen bei der herstellung von proteinmaterialien

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Publication number
EP0116631A1
EP0116631A1 EP83902926A EP83902926A EP0116631A1 EP 0116631 A1 EP0116631 A1 EP 0116631A1 EP 83902926 A EP83902926 A EP 83902926A EP 83902926 A EP83902926 A EP 83902926A EP 0116631 A1 EP0116631 A1 EP 0116631A1
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dna
gene
cells
accordance
cell
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EP0116631A4 (de
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Richard Axel
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Columbia University in the City of New York
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Columbia University in the City of New York
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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
    • 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

Definitions

  • This invention concerns the introduction of DNA which includes a gene or genes coding for, regulating or otherwise influencing the production of proteinaceous materials into eucaryotic cells, that is, cells of organisms classified under the Superkingdom Eucaryotes including organisms of the Plant and Animal Kingdoms. it also concerns the expression of genes which have been introduced into eucaryotic cells. Such genetic intervention is commonly referred to as genetic engineering and in certain aspects involves the use of recombinant DNA technology.
  • the invention disclosed is to be distinguished from the introduction of DNA into organisms of the Superkingdom Procaryotes including particularly bacteria. This distinction is based in part upon the basic differences between eucaryotic and procaryotic cells, the former being characterized by true nuclei formed by nuclear envelopes and by meiosis and the latter being characterized by the absence of well-defined nuclei and the absence of meiosis. Moreover, at the genetic level many genes in eucaryotes are split by non-coding sequences which are not continuously colinear, whereas in .procaryotes, the genes are continuously colinear. Background of the Invention
  • procaryotic organisms particularly bacteria
  • eucaryotic organisms have for the most part proceeded independently of advances in the understanding of eucaryotic organisms, it may be helpful to an appreciation of the present invention to set forth certain developments involving procaryotes.
  • eucaryotic cells specifically mammalian cells, were transformed with foreign DNA coding for a selectable phenotype. Wigler, M. , et al. , Cell 11:223-232 (1977).
  • the present invention has resulted from extending this work. It has been discovered inter alia that eucaryotic cells can be cotransformed to yield transformants having foreign DNA integrated into the chromosomal DNA of the eucaryotic cell nucleus. Moreover, it has unexpectedly been discovered that such foreign DNA can be expressed by the cotransformants to generate functional proteins. In addition, by contrast with procaryotic transformants, the foreign DNA is stably expressed through hundreds of generations, a result that may be attributable to integration of the foreign DNA into the chromosomal DNA.
  • This invention provides major advantages over bacterialsystems for the commercial preparation of proteinaceous materials, particularly proteins of eucaryotic origin such as interferon protein, antibodies, insulin, and the like. Such advantages include the ability to use unaltered genes coding for precursors. After cellular synthesis, the precursor can be further processed or converted within the eucaryotic cell to produce the desired molecule of biological significance. This phenomenon is well known for insulin which is initially produced in the eucaryotic cell as preproinsulin, then converted within the cell to active insulin. Since procaryotic cells lack the requisite cellular machinery for converting preproinsulin to insulin, the insertion into a procaryotic cell of the eucaryotic gene associated with insulin will result in production of preproinsulin, not insulin.
  • interferon is a glycoprotein containing both sugar molecules and protein. If produce in a bacterial cell, the interferon lacks the sugar molecules which are added when interferon is produced in a human cell.
  • proteins produced within bacteria may be contaminated by endotoxins which can cause inflammation if the protein is administered to a mammal without significant purification.
  • interferon produced in a eucaryotic cell would be free of endotoxins. It is therefore another important object of this invention to provide a process for producing compounds which include both non-proteinaceous and proteinaceous moieties such as glycoproteins which cannot be produced in bacterial cells.
  • a foreign DNA I to which an inducible promoter DNA III sequence has been linked can be introduced into a suitable eucaryotic cell by cotransforming the cell with a DNA molecule which includes this combination of foreign DNA I and DNA III and with unlinked DNA II coding for a selectable phenotype not otherwise expressed by the eucaryotic cell.
  • the cotransformation is carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired the selectable phenotype.
  • Proteins and protein-containing products may be produced by cotransforming eucaryotic cells asr described herein and maintaining the cotransformed cells under suitable conditions including the presence of an agent capable of inducing the promoter DNA in sequence so that DNA I is repeatedly transcribed to form mRNAs and the mRNAs so formed are translated to form protein or proteincontaining products.
  • Cotransformation with a DNA molecule which includes foreign DNA I and a promoter DNA III may be combined with gene amplification using as DNA II an amplifiable gene for a dominant selectable phenotype not expressed by the eucaryotic cell.
  • the cotransformation is carried out under suitable conditions including the presence of an antagonist permitting survival or identification of eucaryotic cells which have acquired the dominant selectable phenotype and the presence of an inducing agent for promoter DNA III.
  • FIG. 1 is a schematic flow diagram illustrating the cotransformation process.
  • FIG. 2 is a schematic flow diagram illustrating a proces for recovering foreign DNA I from cotransformed cultured cells using double selection techniques.
  • FIG. 3 Recominant hGH clones, ⁇ 20A is a Charon 4A clone with a 14kb Eco RI fragment. Exons are shown by solid bars. A more detailed map of the sequenced 2.6 kb Eco fragment, "wtGH”, shows the five exons (hatched bars) interrupted by introns A-D.
  • FIG. 4 Recombinant plasmid pGHtk contains an hGH-tk fusion gene. Plasmid pGHtk contains an 0.5 kb Eco RI/Bam HI fragment of hGH (from the 5' end of the 2.6 kb Eco RI fragment) inserted at the Bgl II site of ptk, replacing the tk promoter. Tk information includes the entire coding sequence of the tk gene
  • the initiator ADG is located 50 nucleotides 3' to the Bgl il site.
  • the Bam HI site of the hGH fragment including the putative promoter is 3 nucleotides beyond the transcription initiation site of hGH, De Noto, F.M. , et al. , Nucl. Acids Res. 9:3719-3730 (1981) .
  • Transformation means the process for changing the genotype of a recipient cell mediated by the introduction of purified DNA. Transformation is typically detected by a stable and heritable change in the phenotype of the recipient cell that results from an alteration in either the biochemical or morphological properties of the recipient cell.
  • Cotransformation means the process for carrying out transformations of a recipient cell with more than one different gene.
  • Cotransformation includes both simultaneous and sequential changes in the genotype of a recipient cell mediated by the introduction of DNA corresponding to either unlinked or linked genes.
  • Proteinaceous material means any hiopolymer formed from amino acids.
  • Genotype means the genetic constitution of an organism as distinguished from its physical appearance.
  • Phenotvpe means the observable properties of an organism as produced by the genotype in conjunction with the environment.
  • Selectable phenotype is a phenotype which confers upon an organism the ability to exist under conditions which kill off all organisms not possessing the phenotype. Examples include drug resistance or the ability to synthesize some molecule necessary to cell metabolism in a given growth medium. As used herein, selectable phenotypes also include identifiable phenotypes such as the production of materials which pass from or axe secreted by the cell and can be detected as new phenotypeseither by functional, immunologic or biochemical assays.
  • Interferon protein means the protei naceous part of the glyeoprotein interferon, that is, the portion remaining after removal of the sugar portion.
  • Xt includes the protein portion of interferon derived from human leukocyte, fibroblast or lymphoblastoid cells.
  • Chromosomal DNA means the DNA normally associated with histone in the form of chromosomes residing in the nucleus of a eucaryotic cell.
  • Transcription means the formation of a RNA chain in accordance with the genetic information contained in the DNA.
  • Translation means the process whereby the genetic information in an mSNA molecule directs the order of specific amino acids during protein synthesis.
  • foreign DNA I can be inserted into any eucaryotic cell by cotransforming the cell with DNA I and with unlinked foreign.
  • DNA IT which includes a gene coding for a selectable phenotype not expressed by the cell unless acquired by trans formation. The cotransformation is carried out in a suitable growth medium and in the presence of selective conditions such that the only cells which survive or are otherwise altered axe those which have required the selectable phenotype- See Fig. 1.
  • the present invention is especially useful in connection with the insertion into eucaryotic cells of foreign DNA which includes genes which code for proteinaceous materials not associated with selectable phenotypes. Since such proteinaceous materials are characterized by the fact that they are not associated with a selectable phenotype, cells which contain DNA coding therefore cannot be identified exceptby destruction of the transformed cell and examination of its contents. Examples of proteinacecus materials, the genes for which may be inserted into and expressed by eucaryotic cells using the cotransformation process include interferon protein, insul in, growth hormones, clotting factors, viral antigens, enrymes and antibodies.
  • DNA I and DNA II may not need to be purified to obtain integration and expression, it is oftentimes preferable that the DNAs be purified prior to use in cotransforming cells. Such purification I imi ts the possibility of spurious results due to the presence of contaminants and increases the probability that cotransformed cells can be identified and stably cultured.
  • DNA I and/or DNA II have been obtained by restriction endonuclease cleavage of chromosomal donor DNAs . such as , for example, restriction endonuclease cleavage of encaryotic chrom ⁇ somal DNA.
  • DNA I and DNA II be treated with calciumr phosphate prior to use in cotransformi ng eucaryatic cells.
  • the procedure for so treating DNA with calcium phosphate is set forth more fully hereinafter.Final ly, it is preferable that the foreign DNA I be present during cotransformation in an amount relative to DNA II coding for a selectable phenotype which constitutes an excess of the former, such as an amount in the range from about 1:1 to about 100 , 000 :1.
  • the foreign DNA I and/or the foreign DNA II are attached to bacterial plasmid or phage DNA prior to use in cotransforming eucaryotic cells .
  • foreign DNA X and/or DNA IX are attached to phage DNA and then encapsidated in phage particles prior to cotransformation.
  • any DNA II coding for a selectable phenotype would be useful in the co transformation process of the present invention, the experimental details set forth particularly concern the use of a gene for thymidine kinase obtained from herpes simplex virus and the use of a gene for adenine phosphoribosyl tran sferase .
  • a DNA II which includes a gene coding for a selectable phenotype associated with drug resistance, e.g. , a mutant dihydrofolate reductase gene which renders cells resistant to methotrexate greatly extends the applicability of the process .
  • the co- transformation involves DNA I which is physically and chemically unlinked to DNA II, and the DNA I is stably int egrated into the chromosomal DNA within the nucleus of the co trans formed eucaryotic cell.
  • Cotransformation in accordance with this invention may be carried out in any suitable medium, limited. only in that cotransformed cells be capable of survival and/or identification on the medium.
  • a suitable medium for mouse fibroblast cells which have acquired the thymidine kinase gene is HAT described more fully hereinafter.
  • the cotransformation is carried out in the presence of selective conditions which permit survival and/or identification of those cells which have acquired the selectable phenotype. Such conditions may include the presence of nutrients, drug or other chemical antagonists, temperature and the like.
  • Eucaryotic cells cotransformed in accordance with this invention contain foreign DNA I coding for desired materials which can be recovered from the cells using technfques well known in the art.
  • the cells can he permitted to transcribe DNA I to form mRNA which in turn is translated to form protein or other desired material which may be recovered, again using well known techniques - Finally , the cells can be grown in culture, harvested and protein or other desired material recovered therefrom.
  • the process can be equally useful in the production of synthetic biopolymers for which synthetic genes are constructed.
  • the instast invention provides a process for producing novel proteins net yet in existence. Additionally, it provides a process. for producing proteins which , althoagh they presently exist, da so is such minute quantities or in such impure form that their isolation and/car ident ification cannot otherwise be effected. Finally, the inventios provides a process fo ⁇ producing partially proteinaceous products such as the glycoprotains and other products , the synthesis of which is genetically directed.
  • Another aspect of the invention involves processes for inserting multiple copies of genes into eucaryotic cells in order to increase the amount of gene product formed within the cell .
  • One process for ins erting a multiplicity of foreign DNA I molecules into a eucaryotic cell comprises cotransforming the cell with multiple DNA I molecules and with multiple, unlinked foreign DNA II molecules corresponding to multiple copies of an amplifiable gene for a dominant selectable phenotype not otherwise expressed by the cell .
  • This cotransformation process is carried out in a suitable medi um and in the presence of an agent permitting survival and/or identiffcafion of cel Is which acquire the dominant selectable phenotype.
  • this is done in the presence of successively higher concentrations of such an agent so that only those cells acquiring the highest number of amplifiable dominant genes CDNA II) survive and/or are identified.
  • Cotransformed eucaryotic cells which have acquired multinle copies of DNA I may then be used to produce increased amounts of the gene product for wh i ch DNA I codes in the same manner as described hereinabove.
  • multiple copies of foreign genes can be generated in and ultimately expressed by eucaryotic cells by transforming the eucaryotic cells with DNA molecules, each of which has been formed by linking a foreign DNA I to a foreign DNA II which corresponds to an amplifiable gene for a dominant selectable phenotype not normally expressed by the eucaryotic cell.
  • the linkage between DNA I and DNA II is preferably in the form of a chemical bond, particularly a bond formed as a result of enzymatic treatment with a ligase.
  • Transformation with such hybrid DNA molecules so formed is then carried out in a suitable growth medium and in the presence of successively elevated concentrations , e.g., amounts ranging from 1:1 to 10,000:1 on a molariry basis, of an agent which permits survival and/or identi fication of those eucaryotic cells which have acquired a sufficiently high number of copies of the amplifiable gene.
  • concentrations e.g., amounts ranging from 1:1 to 10,000:1 on a molariry basis
  • genes associated with drug resistance e.g., the gene for dihydrofblate reductase which renders cells resistant to methotrexate, are particularly suitable.
  • waltiple copies of proteinaceous or other desired molecules can be produced within eucaryotic cells.
  • multiple molecules of interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody or of interferon per se can be produced by eucaryotic cells, particularly mammalian cells, which have been transformed using hybrid DNA or cotransformed using purified DNA which has been treated with calcium phosphate in the manner described hereinafter.
  • this invention provides a process for producing highly desired, rare and costly proteinaceous and other biological materials in concentrations not obtainble using conventional techniques.
  • Still another aspect of the present invention involves the preparation of materials normally produced within eucaryotic cells in minute amounts such as glycoproteins including interferon, which are in part protein but additionally include other chemical species such as sugars, ribonucleic acids, histones and the like.
  • materials normally produced within eucaryotic cells such as glycoproteins including interferon, which are in part protein but additionally include other chemical species such as sugars, ribonucleic acids, histones and the like.
  • a gene or genes for the protein portion of a cellular material such as a glyeoprotein, which includes a non-protein portion
  • a cellular material such as a glyeoprotein, which includes a non-protein portion
  • the cell will not only produce the corresponding proteinaceous material but will utilize already existing cellular mechanisms to process the proteinaceous materials, if and to the extent necessary, and will also add the appropriate non-proteinaceous material to form the complete, biologically active material.
  • the complete biologically active glyprotein, interferon could be prepared by first synthesizing interferon protein in the manner described and additionally permitting the cell to produce the non-proteinaceous or sugar portion of interferon and to synthesize or assemble true interferon therefrom.
  • the interferon so prepared could then be recovered using conventional techniques.
  • eucaryotic cells have been stably transformed with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist.
  • the addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT medium. Since these biochemical transformants might represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population; cotransformation experiments were performed with the viral tk gene and bacteriophage ⁇ X174, plasmid pBR 322 or cloned chromosomal human or rabbit ⁇ -globin gene sequences.
  • Tk transformants were cloned and analyzed for cotransfer of additional DNA sequences by blot hybridization. In this manner, mouse cell lines were identified which contain multiple copies of ⁇ X, pBR 322, or human and rabbit ⁇ -globin sequences. From one to more than 50 cotransformed sequences are integrated into high molecular weight DNA isolated from independent clones. Analysis of subclones demonstrates that the cotransformed DNA is stable through many generations in culture. This cotransformation system allows the introduction and stable integration of virtually any defined gene into cultured eucaryotic cells. Ligation to either viral vectors or selectable biochemical markers is not required.
  • Cotransformation with dominant-acting markers should in principle permit the introduction of virtually any cloned genetic element into wild-type cultured eucaryotic cells.
  • a dominant-acting, methotrexate resistant, dihydrofolate reducatse gene from CHO A29 cells was transferred to wild-type cultured mouse cells.
  • definitive evidence for gene transfer was provided. Exposure of these cells to elevated levels of methotrexate results in enhanced resistance to this drug, accompanied by amplification of the newly transferred gene.
  • the mutant DHFR gene therefore, has been used as a eucaryotic vector, by ligating CHO A29 cell DNA to pBR 322 sequences prior to transformation.
  • Amplification of the DHFR sequences results in amplification of the pBR 322 sequences.
  • the use of this gene as a dominant-acting vector in eucaryotic cells will expand the repetoire of potentially transformable cells, no longer restricting these sort of studies to available mutants.
  • the cloned chromosomal rabbit ⁇ -globin gene has been introduced into mouse fibroblasts by DNA-mediated gene transfer.
  • the cotransformed mouse fibroblast containing this gene provides a unique opportunity to study the expression and subsequent processing of these sequences in a hetero logous host.
  • Solution hybridization experiments in concert with RNA blotting techniques indicate that in at least one transformed cell line rabbit globin sequences are expressed in the cytoplasm as a polyadenylated 9S species. These 9S sequences result from perfect splicing and removal of the two intervening sequences.
  • the aprt gene of the chicken is not cleaved by the enzyme, Hin III or Xba , and transformation of aprt- mouse cells with cellular DNA digested with these enzymes results in the generation of aprt + clonies which express the chicken aprt genes .
  • Ligation of Sin Ill-cleaved chicken DNA with Hin III-cleaved plasmid pBR 322 results in the formation of hybrid DNA molecules in which the aprt gene is now adjacent to plasmid sequences . Trans formation of aprt- cells is now performed with this DNA.
  • ransformants should contain the aprt gene covalentlv linked to pBR 322 with this entire complex integrated into high molecular weight DNA in the mouse cell .
  • This initial cellular transformation serves to remove the chicken aprt gene from the vast majority of other chick sequences .
  • This transformed cell DNA is now treated with an enzyme , Xba I , which does not cleave either pBR 322 or the aprt gene .
  • the resultant fragments are then circularized with iigase
  • One such fragment should contain the aprt gene covalently linked to pBR 322 sequences coding for an origin of replication and the ampicillin resistance murker. Transformation of a bacterium such as E .
  • coli with these circular markers selects for plasmid sequences from eucaryotic DNA which are now linked to chicken aprt sequences.
  • This double selection technique should permit the isolation of genes expressed at low levels in eucaryotic cells for which hybridization probes are not readily obtained.
  • tk- Friend cell is extremely refractory to transformation with a cloned viral tk gene and can only be transformed at levels from 10 -5 to 10 -6 , the frequency observed in mouse L cells. Nonetheless, this difficulty has been overcome and numerous tk + transformants have been obtained which retain and express the donor viral gene.
  • a series of cotransformation experintents have been performed with wild type chromosomal clones containing adult human globin genes and 5-10 kb of 5' and 3' flanking information.
  • a series of mouse cotransformants have been identified which now contain 1-5 copies of tfte human ⁇ globin gene. A series of experiments were carried out to determine whether this human gene is expressed after induction in these murine cells.
  • a.promoter DNA sequence for the globin gene which, in the presence of DMSO, mediates the increased synthesis of RNA coding for globin.
  • a desired gene such as a gene coding for insulin, interferon protein or the like may be used in the cotransformation of a eucaryotic cell after being linked with the promoter sequence. Cotransformed cells will then produce in the presence of DMSO markedly elevated levels of the protein for which the gene codes.
  • the promoter sequence, joined to a desired gene, e.g. , for an antibody may be used in cotransformation with an amplifiable gene for drug resistance.
  • the cotransformed cells may then be cultured both in the presence of successively elevated concentrations of the appropriate drug and in the presence of DMSO to produce high levels of proteinaceous material.
  • inducible promoter sequence involves the human growth hormone gene.
  • the human growth hormone gene in pituitary cells is tightly controlled by levels of thyroid hormone and corticosteroid.
  • the addition of these hormones results in a dramatic inductive effect which at least in part is reflected by an enormous increase in the appearance of translatable mRNA.
  • the molecular mechanisms responsible for hormonal induction remain obscure.
  • Tentative evidence suggests that induction results at least in part from the interaction of specific hormone receptor complexes with the chromosome which may result in transcriptional activation. Strong evidence in support of this attractive hypothesis, however , is lacking at present. Transformation may provide the means to discern whether specific sequences reside close to structural genes so as to render these genes responsive to hormone induction.
  • Rat pituitary cells, GH-3 synthesize increasing amounts of rat growth hormone in response to the addition of corticosteroids or thyroid hormone. Therefore, one may introduce the cloned human growth hormone gene into these cells and place it under hormonal control in its new cellular environment. One may then begin to alter this gene, creating a series of deletions of DNA from the 5' and 3' termini of the transcript to discern whether hormonal induction is maintained in these deletion mutants. In this way, one may localize potential sequences which render a gene responsive to hormone action.
  • sequences may then be genetically transplanted to appropriate loci on other genes such as globin genes and in this way definitively prove the regulatory effect of such sequences on gene expression.
  • this system provides a promoter which is inducible in the presence of hormone and can be used in cotransformation, with or without amplification, in the manner described hereinabove.
  • cotransformation provides an approach to genetic therapy. Specifically, one may therapeutically treat and perhaps cure genetically defective eucaryotic cells in order to alleviate associated symptoms by cotransforming the defective cells with DNA which includes a gene for a selectable marker and with DNA which includes a genetically correct gene.
  • the defective cells may then be cotransformed with a competent globin gene and with a gene conferring drug resistance being used as a marker.
  • the cotransformation is carried out in the presence of the drug.
  • the cotransformed cells may then be transplanted back into the patient, and the patient maintained on dosages of the drug such that only transformed cells survive. Since the cells will be identical in all respects except for the additional gene, the transplanted cells should not be rejected as is the case with transplants using foreign cells. This approach may lead to a cure for sickle cell anemia and for other genetically-based illnesses.
  • thymidine kinase (tk) gene from herpes simplex virus to mutant mouse cells lacking tk results in the appearance of stable transformants expressing the viral gene which can be selected by their ability to grow in HAT.
  • tk thymidine kinase
  • ⁇ X174 DNA was initially used in cotransformation experiments with the tk gene as the selectable marker .
  • ⁇ X replicative form DNA was cleaved with Pst 1 , which recognizes a single site in the circular genome .
  • Sanger, F . et al . Nature 265: 687-695 (1977) .
  • 500 pg of the purified tk gene were mixed with 1-10 ug of Pst-cleaved ⁇ x replica tive form DNA. This DNA was. then added to mouse Ltk- cells using the transformation conditions described under Methods and Materials h.er einaf ter.
  • tk transformant After 2 weeks in selective medium (HAT) , tk transformant were observed at a frequency of one colony per 10 cells per 20 Pg of purified gene. Clones were picked and grown to mass culture. It was then asked whether tk transfo ⁇ mants also containe
  • Sco RI the number of bands observed reflects the minimum number of eucaryotic DNA fragments containing information homologous to ⁇ X.
  • the clones contain variable amounts of ⁇ X sequences .
  • Clones ⁇ X1 and ⁇ X2 reveal a single annealing fragment which is smaller than the ⁇ X genome. In these clones , therefore, only a portion of the transforming sequences persist. There was also observed a tk - transfcrmant (.clone ⁇ X3) with no detectable ⁇ X sequences .
  • Clones ⁇ X4 , 5 , 6 , and 7 reveal numerousr high molecular weight bands which are too closely spaced to count, indicating that th.ese clones contain multiple ⁇ X-specific fragments. These experiments demonstrate cotransformation of cultured mammalian cells with the viral tk gene and ⁇ X DNA.
  • the clones contained varying amounts of ⁇ X DNA .
  • none of fifteen clones picked at random from neutral medium contained any ⁇ X information.
  • the addition of a selectable marker facilitates the identification cf those cells which contain ⁇ X DNA.
  • Cleavage of DNA from ⁇ X transformants with Sco RI generates a series of fragments which contain ⁇ X DNA sequences . Th.e ⁇ e fragments may reflect multiple integration events . Alternatively , thes e fragments could result from tandem arrays of complete or partial ⁇ X sequences which are not integrated into cellular DNA. To distinguish between these possibilities, transformed cell DNA was cut with BAM HI or Eco RI, neither of which cleaves the ⁇ X genome. If the ⁇ X DNA sequences were not integrated, neither of these enzymes would cleave the ⁇ X fragments. If the ⁇ X DNA sequences were not integrated, neither of these enzymes would cleave the ⁇ X fragments. If the ⁇ X DNA sequences were not integrated, neither of these enzymes would cleave the ⁇ X fragments.
  • the location of ⁇ X sequences in transformed cells was determined by subcellular fractionation. Nuclear and cytoplasmic fractions was prepared, and the ⁇ X DNA sequence content of each was assayed by blot hybridization. The data indicate that 95% of the ⁇ X sequences are located in the nucleus. High and low molecular weight nuclear DNA was prepared by Hirt fractionation. Hirt, B. J. , Mol. Biol. 26: 365-369 (1967) . Hybridization with DNA from these two fractions indicates that more than 95% of the ⁇ X information co-purifies with the high molecular weight DNA fraction. The small amount of hybridization observed in the supernatant fraction reveals a profile identical to that of the high molecular weight DNA, suggesting contamination of this fraction with high molecular weight DNA.
  • annealing profiles of DNA from transformed clones digested wtih enzymes that do not cleave the ⁇ X genome provide evidence that integration of ⁇ X sequences has occurred and allow us to estimate the number of ⁇ X sequences integrated.
  • Annealing profiles of DNA from transformed clones digested with enzymes which cleave within the ⁇ X genome allow us to determine what proportion of the genome is present and how these sequences are arranged following integration.
  • Cleavage of ⁇ X with the enzyme Hpa I generates three fragments for each integration event: two "internal" fragments of 3.7 and 1.3 kb which together comprise 90% of the ⁇ X genome, and one "bridge" fragment of 0.5 kb which spans the Pst I cleavage site.
  • the annealing pattern of clone 5 DNA cleaved with Hpa I is more complex. If internal fragments are present, they are markedly reduced in intensity; instead, multiple bands of varying molecular weight are observed. The 0.5 kb Hpa I fragment which bridges the Pst 1 cleavage site is not observed for either clone ⁇ X4 or clone ⁇ X5.
  • the annealing pattern obser ved for four of the six ⁇ X4 subclones is virtually identical to that of the parent. In two subclones , an additional Eco RI fragment appeared which is of identical molecular weight in both .
  • pBR322 linearized with BAM HI was mixed with the purified viral tk gene in a molar ratio of 1000:1. Tk + transformants were selected and scored for the presence of pBR322 sequences. Cleavage of BAM HI linearized pBR322 DNA with Bgl I generates two internal fragments of 2.4 and 0.3 kb. The sequence content of the pBR322 transformants was determined by digestion of transformed cell DNA with Bgl I followed by annealing with 32 P-labeled plasmid DNA. Four of five clones screened contained the 2.4 kb internal fragment. The 0.3 kb fragment would not be detected on these gels.
  • Transformation with purified eucaryotic genes may provide a means for studying the expression of cloned genes in a heterologous host. Cotransformation experiments were therefore performed with the rabbit major globin gene which was isolated from a cloned library of rabbit chromosomal DNA (Maniatis, T., et al., Cell 15: 687-701
  • R ⁇ G-1 One ⁇ -globin clone designated R ⁇ G-1 consists of a 15 kb rabbit DNA fragment carried on the bacteriophage cloning vector Charon 4a .
  • Intact DNA from this clone (R ⁇ G-1 ) was mixed with the viral tk DNA at a mola r ratio of 100 : 1 , and tk + trans formants were isolated and examined for the presence of. rabbit globin sequences .
  • Cleavage of R ⁇ G-1 with the enzyme Kpn I generates a 4 .7 kb fragment which contains the entire rabbit ⁇ -globin gene . This fragment was purified by gel electrophoresis and nick translated to generate a probe for subsequent annealing experiments .
  • the ⁇ -globin genes of mouse and rabbit are partially homologous , although we do not observe annealing of the rabbit ⁇ -globxn probe with Kpn-cleaved mouse DNA under our experimental conditions. In contrast, cleavage of rabbit liver DNA with Kpn I generates the expected 4.7 kb globin band. Cleavage of transformed cell DNA with the enzyme Kpn I generates a 4.7 kb fragment containing globin-specific information in six of the eight tk + transformants examined. In two of the clones, additional rabbit globin bands are observed which probably result from the loss of at least one of the Kpn sites during transformation. The number of rabbit globin genes integrated in these transformants is varidile.
  • A29 Mtx RIII contains a mutation in the structural gene for dihydro folate reductase, reducing the affinity of this enzyme for methotrexate. Flintoff , W. F. et al . , Somatic Cell Genetic 2% 245-261 (1976) . Genoroic DNA from this line was used to transform clones ⁇ X4 and ⁇ X5 and Ltk- cells . The frequency of trans formation to mtx resistance for the ⁇ X clones was identical to that observed with the parental Ltk- cells . It is therefore concluded that competence is not a stably heritable trait and may be a transient property of cells .
  • biochemical transformants will represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population.
  • cultures were cotransformed with a physically unlinked gene which provided a selectable marker. This cotransformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells . Ligation to either viral vectors or selectable biochemical markers is not required.
  • At least one of the rabbit ⁇ -globin mouse transformants expresses polyadenylat ⁇ d rabbit ⁇ -globin RNA sequences as a discrete 9S cytoplasmic species .
  • the elaborate processing events required to generate 9S globin RNA correctly are unlikely to occur in procaryotes .
  • the ⁇ X cotransformants were studied in greatest detail. The frequency of cotransformation is high: 14 of 16 tk + transformants contain ⁇ X sequences. The ⁇ X sequences are integrated into high molecular weight nuclear DNA.
  • the number of integration events varies from one to more than fifty in independent clones.
  • the extent of the bacteriophage genome present within a given transformant is also variable; while some clones have lost up to half the genome, other clones contain over 90% of the ⁇ X sequences. Analysis of subclones demonstrates that the ⁇ X genotype is stable through many generations in culture. Similar conclusions are emerging from the characterization of the pBR322 and globin gene cotransformants.
  • Hybridization analysis of restriction endonuclease-cleaved transformed cell DNA allows one to make some preliminary statements on the nature of the integration intermediate. Only two ⁇ X clones have been examined in detail . In both clones , the donor DNA was Pst I-linearized ⁇ X DNA.
  • Competent cells may be genetic variants within the culture ; however , these studies indicate that the competent phenotype is not stably inherited. If one can extrapolate from studies in procaryotes , the phenomenon of competence is likely to be a complex and transient property reflecting the metabolic state of the call .
  • Cotransformants contain at least one copy of the tx gene and variable amounts of ⁇ X DNA. Although transformation was performed with ⁇ X and tk sequences at a molar ratio of 1000:1, the sequence ratio observed in the transformants never exceeded 100:1. There may be an upper limit to the number of integration events that a cell can tolerate, beyond which lethal mutations occur. Alternatively, it is possible that the efficiency of transformation may depend upon the nature of the transforming fragment. The tk gene may therefore represent a more efficient transforming agent than phage DNA.
  • Cotransformed mouse fibroblasts containing the rabbi t ⁇ -globin gene provide an opportunity to study the expression and subsequent process ing of these sequences in a heterologous host.
  • the purified tk gene was mixed with a 100-fold molar excess of intact recombinant DNA from clone R ⁇ Gl . This DNA was then exposed to mouse Ltk- cells under transformation conditions described herein under Methods and Materials . After 2 weeks in selective medium , tk trans formants were observed at a frequency of one colony per 10 6 cells per 20 pg of tk gene . Clones were picked and grown into mass cul ture .
  • the number of rabbit globin genes integrated in these transformants was variable: some clones contained a single copy of the gene, whereas others contained up to 20 copies of the heterologous gene. It should be noted that the ⁇ -globin genes of mouse and rabbit are partially homologous . However, we do not observe hybridization of the rabbit ⁇ -globin probe to Kpn-cleaved mouse DNA , presumably because Kpn cleaveage of mouse DNA leaves the ⁇ -gene cluster in exceedingly high molecular weight fragments not readily detected in these experiments . These results demonstrate the introduction of the cloned chromosomal rabbit ⁇ -globin transfer.
  • the cotransformation system may provide a functional assay for cloned eucaryotic genes if these genes are expressed in the ⁇ eterolog ⁇ us recipient cell.
  • Six transformed cell clones were therefore analyzed for the presence of rabbit ⁇ -globin RNA sequences.
  • solution hybridization reactions were performed to determine the cellular concentration of rabbit globin transcripts in our transformants.
  • a radioactive cDNA copy of purified rabbit ⁇ - and ⁇ -globin mRNA was annealed with the vast excess of cellular RNA.
  • This rabbit globin cDNA was used as a probe in hybridization reactions with total RNA isolated from six transformed cell lines.
  • Total RNA from transformed clone ⁇ protected 44% of the rabbit cDNA at completion, the value expected if only ⁇ -gene transcripts were present.
  • This reaction displayed pseudo- first-order kinetics with R 0 t 1/2 of 2 x 10 3 .
  • a second transformant reacted with an R 0 t 1 / 2 of 8 x 10 3 . No significant hybridization was observed at R 0 ts _ 10 4 with total RNA preparations from the four additional transformants.
  • RNA from clcne 6 was fractionated into nuclear and cvtoolasmic populations to determine the in tracellular localization of the rabbit globin RNA.
  • the cytoplasmic RNA was further fractionated by oligo (dT) -cellulose chromatography into poly (A) + and poly (A) - RNA.
  • Poly (A) + cytoplasmic RNA from clone 6 hybridizes with the rabbit cDNA with an
  • the steady-state concentration of rabbit ⁇ -globin RNA present in our transformant can be calculated from the R 0 t 1 / 2 to be about five copies per cell , with greater than 90 % localized in the cytoplasm.
  • cDNA was prepared from purified 9S mouse globin RNA. This cDNA does not hybridize with poly (A) RNA from clone 6 at R 0 t values at which the reaction with rabbit globin cDNA is complete (ii) Rabbit globin cDNA does not hybridize with total cellular RNA obtained with tk + globin- transformants at R 0 t vlaues exceeding 10 4 .
  • the ⁇ -globin gene sequences are detected as a 14S precursor RNA that reflects transcription of two intervening sequences that are subsequently removed from this molecule to generate a 9S messenger RNA. It was therefore of interest to determine whether the globin transcripts detected exist at a discrete 9S species , which is likely to reflect appropriate splicing of the rabbit gene transcript by the mouse fibroblast.
  • Cytoplasmic poly (A) -containing RNA from clone 6 was electrophoresed on a methyl-mercury/agarose gel, Bailey, J. & Davidson, N. , Anal. Biochem. 29 : 75-85 (1976) , and transferred to diazotized cellulose paper.
  • RNA on the filters was hybridized with DNA from the plasmid p ⁇ Gl , which contains rabbit ⁇ -globin cDNA sequences .
  • a discrete 9S species of RNA was observed in the cytoplasm of the transformant, which comigrated with rabbit globin mRNA isolated from rabbit ery throb lasts .
  • Hybridization to 9S RNA species was not observed in parallel lanes containing either purified mouse 9S globin RNA or poly (A) -containing cytoplasmic RNA from a tk transformant containing no rabbit globin genes .
  • DNA of the ⁇ clone, R ⁇ GI containing the chromosomal rabbit ⁇ - globin sequence hybridized with transformed fibroblast RNA.
  • the hybrid formed was treated with SI nuclease, and the protected DNA fragments were analyzed by alkaline agarose gel electrophoresis and identified by Southern blotting procedures. Southern, E. M. , J. Mol. Biol. 98: 503-517 (1975) .
  • the hybridization of mature rabbit mRNA to R ⁇ Gl DNA generates three DNA fragments in this sort of analysis: a 146-base pair fragment spanning the 5' terminus to the junction of the small intervening sequence, a 222-base pair internal fragment bridging the small and large intervening sequences, and a 221-base pair fragment spanning the 3' junction of the large intervening sequence to the 3' terminus of the mRNA molecule.
  • transformant RNA was analyzed in this fashion, a 222-base pair fragment was observed as well as an aberrant fragment of 100 base pairs but no 146-base pair fragment.
  • mice cell lines have been constructed that contain the rabbit ⁇ -globin gene.
  • the ability of the mouse fibroblast recipient to transcribe and process this heterologous gene has then been analyzed.
  • Solution hybridization experiments in concert with RNA blotting techniques indicate that, in at least one transformed cell line, rabbit globin sequences are expressed in the cytoplasm as a polyadenylylated 9S species. Correct processing of the rabbit ⁇ -globin gene has also been observed in tk mouse cell transformants in which the globin and tk plasmids have been ligated prior to transformation. Mantei, N. , et al., Nature (London) 281: 40-46 (1970).
  • 45 nucleotides present at the 5' terminus of mature rabbit mRNA are absent from the ⁇ -globin RNA sequence detected in the cytoplasm of the transformant examined. It is possible that incorrect initiation of transcription occurs about the globin gene in this mouse cell line.
  • the globin sequences detected may result from transcription of a long precursor that ultimately must undergo 5 ' processing to generate the mature 9S species. Incorrect processing at the 5' terminus in the mouse fibroblast could be responsible for the results. At present, it is difficult to distinguish among these alternatives.
  • globin sequences in transformed fibroblasts. It is possible that constitutive synthesis of globin RNA occurs in cultured fibroblasts, Humphries, S., et al., Cell 7: 267-277 (1976), at levels five to six orders of magnitude below the level observed in erythroblasts. The introduction of 20 additional globin DNA templates may simply increase this constitutive transcription to the levels observed in the transformant. Alternatively, it is possible that the homologous globin aene is repressed by factors that are partially overcome by a gene dosage effect provided by the introduction of 20 additional globin genes.
  • normal repression of the globin gene in a fibroblast may depend upon the position of these sequences in the chromosome. At least some of the newly introduced genes are likely to reside at loci distant from the resident mouse globin genes. Some of these ectopic sites may support low level transcription. Present data do no permit one to distinguish among these and other alternatives.
  • RNA 9S globin RNA in the cytoplasm of transformants suggests that this RNA may be translated to give rabbit ⁇ -globin polypeptide. Attempts to detect this protein in cell lysates using a purified anti-rabbit B-globin antibody have thus far been unsuccessful. It is possible that the globin RNAs in the transformant are not translated or are translated with very low efficiency due to the ab sence of a functional ribosomal binding site.
  • the cytoplasmic globin transcripts in the transformant lack about 48 nucleotides of untranslated 5' sequence, which includes 13 nucleotides known to interact with the 40S ribosomal subunit in nuclease protection studies.
  • ⁇ X DNA was used in cotransformation experiments with the tk gene as the selectable marker.
  • ⁇ X replicative form DNA was cleaved with Pst I, which recognizes a single site in the circular genome, Sanger, F. etal., Nature 265: 687-695 (1977).
  • Purified tk gene 500 pg was mixed with 1-10 ug of Pst-cleaved ⁇ X replicative form DNA. This DNA was then added to mouse Ltk cells using the transformation conditions described herein and in Wigler, M. , et al., Cell 16: 777-785 (1979). After two weeks in selective medium
  • HAT HAT
  • tk + transformants contained ⁇ X DNA sequences.
  • High molecular weight DNA from the transformants was cleaved with the restriction endo nuclease Eco RI, which recognizes no sites in the ⁇ X genome.
  • the DNA was fractionated by agarose gel electro phoresis and transferred to nitrocellulose filters, and these filters were then annealed with nick-translated 3 2 P- ⁇ X DNA (blot hybridization).
  • Transformation with purified eucaryotic genes provides a means for studying the expression of cloned genes in a heterologous host. Cotransformation experiments were performed with the rabbit ⁇ major globin gene which was iso lated from a cloned library of rabbit chromosomal DNA.
  • R G-1 One ⁇ -globin clone, designated R G-1 consists of a 15 kb rabbit DNA fragment carried on the bacteriophage ⁇ cloning vector Charon 4A. Intact DNA from this clone (kpG-1) was mixed with the viral tk DNA at a molar ratio of 100:1, and tk transformants were isolated and examined for the presence of rabbit globin sequences. Cleavage of R ⁇ G-1 with the enzyme Kpn I generates a 4.7 kb fragment which contains the entire rabbit ⁇ -globin gene. This fragment was purified by gel electrophoresis and nick-translated to generate a probe for subsequent annealing experiments.
  • the ⁇ -globin genes of mouse and rabbit are partially homologous, although we do not observe annealing of the rabbit ⁇ -globin probe with Kpn-cleaved mouse DNA, presumably because Kpn generates very large globin-specific fragments.
  • cleavage of rabbit liver DNA with Kpn I generates the expected 4.7 kb globin band.
  • Cleavage of transformed cell DNA with the enzyme Kpn I generates a 4.7 kb fragment containing globinspecific information in six of the eight tk transformants examined.
  • the number of rabbit globin genes present in these transformants is variable. In comparison with controls, some of the clones contain a single copy of the gene, while others may contain as many as 20 copies of this heterologous gene.
  • the cotransformation system developed provides a functional assay for cloned eucaryotic genes if these genes are express in the heterologous recipient cell.
  • Six transformed cell clones were analyzed for the presence of rabbit -globin RNA sequences.
  • solution hybridization reactions were performed to determine the cellular concentration of rabbit globin transcripts in transformants.
  • the ⁇ -globin gene sequences are detected as a 14S precursor RNA which reflects transcription of two intervening sequences which are subsequently spliced from this molecule to generate a 9S messenger RNA.
  • Our solution hybridization experiments only indicate that polyadenylated rabbit globin RNA sequences are present in the mouse transformant. It was therefore of interest to determine whether the globin transcripts we detected exist as a discrete 9S species, which is likely to reflect appropriate splicing of the rabbit gene transcript by the mouse fibroblast.
  • Cytoplasmic poly A-containing RNA from clone 6 was denatured by treatment with 6M urea at 70°C, and electrophoresed on a 1% acid-urea-agarose gel and transferred to diazotized cellulose paper. Following transfer, the RNA filters were hybridized with DNA from the plasmid R ⁇ G-1 containing rabbit ⁇ -globin cDNA sequences.
  • Linearized pBR 322 DNA is introduced into mouse Ltk- cells via cotransformation using the tk gene as a selectable marker. DNA is isolated from transformants and screened for the presence of pBR 322 sequences. Since the donor plasmid is linearized, interrupting the tetracycline resistant gene, transformed cell DNA contains a linear stretch of plasmid DNA consisting of the replication origin and the ⁇ -lactamase gene covalently linked to mouse cellular DNA. This DNA is cleaved with an enzyme such as Xho I, which does not digest the plasmid genome. The resulting fragments are circularized at low DNA concentrations in the presence of ligase. Circular molecules containing plasmid DNA are selected from the vast excess of eucaryotic circles by transformation of E. coli strain ⁇ l776.
  • the frequency with which DNA is stably introduced into competent cells is high. Furthermore, the cotransformed sequences appear to be integrated into high molecular weight nuclear DNA. The number of integration events varies from one to greater than fifty in independent transformed clones. At present, precise statements cannot be made concerning the nature of the integration intermediate. Although data with ⁇ X are in accord with the model in which ⁇ X DNA integrates as a linear molecule, it is possible that more complex intramolecular recombination events generating circular intermediates may have occurred prior to or during the integration process. Whatever the mode of integration, it appears that cells can be stably transformed with long stretches of donor DNA. It has been observed that transformants contain contiguous stretches of donor DNA 50 kb long. Furthermore, the frequency of competent cells in culture is also high. At least one percent of the mouse Ltk cell recipients can be transformed to the tk + pheno type. Although the frequency of transformation in nature is not known, this process could have profound physiologic and evolutionary consequences.
  • mouse fibroblast contains the enzymes necessary to transcribe and correctly process a rabbit gene whose expresseion is normally restricted to erythroid cells. Similar observations have been made by others using a viral vector to introduce the rabbit globin gene into monkey cells.
  • Mtx resistant cell lines have been identified which fall into three categories:
  • A29 An interesting methotrexate resistant variant cell line (A29) has been identified that synthesizes elevated levels of a mutant dihydrofolate reductase with reduced affinity for methotrexate. Wigler, M. , et al.. Cell 16:777-785 (1979). Genomic DNA from this cell line has been used as donor in experiments to transfer the mutant dhfr gene to mtx sensitive cells. Exposure of mtx resistant transformed cells to increasing levels of mtx selects for cells which have amplified the transferred gene. In this way, it is possible to trans fer and amplify virtually any genetic element in cultured mammalian cells.
  • High molecular weight cellular DNA was prepared from wildtype mtx sensitive CHO cells and from A29 cells, an mtx resistant CHO derivative synthesizing increased levels of a mutant dhfr. Flintoff, W. F. , et al.. Cell 2:245-262 (1976).
  • the ability of these DNA preparations to transfer either the dhfr gene or the tk gene to tk mouse L cells (Ltk- aprt-) was tested using a modification of the calcium phosphate coprecipitation method. Wigler, M. , et al., Proc. Nat. Acad. Sci . USA 76:1373-1376 (1979).
  • DNA from both mutant A29 and wild-type CHO cells was competent in transferring the tk gene to Ltk- aprt- cells. Methotrexate resistant colonies were observed only following treatment of cells with DNA from A29. The data obtained suggest that treatment of methotrexate sensitive cells with A29 DNA resulted in the transfer and expression of a mutant dhfr gene, thus rendering these cells insensitive to elevated levels of methotrexate.
  • DNA was cleaved with restriction endonuclease Hind III, electrophoresed in agarose gels, and transferred to nitrocellulose filters. These filters were then hybridized with high specific activity, 32P-labeled nick-translated pdhfr-21 and developed by autoradiography. This procedure visualizes restriction fragments of genomic DNA homologous to the dhfr probe. Prominent bands are observed at 15 kb, 3.5 kb and 3 kb for mouse DNA and 17 kb, 7.9 kb, 3.7 kb and 1.4 kb for hamster DNA. The restriction profiles between these two species are sufficiently different to permit one to distinguish the hamster gene in the presence of an endogenous mouse gene.
  • Selectable genes can be used as vectors for the introduction of other genetic elements into cultured cells. In previous studies, it has been demonstrated that cells transformed with the tk gene are likely to incorporate other unlinked genes. Wigler, M. , et al., Cell 16:777-785 (1979). The generality of this approach was tested for the selectable marker, the mutant dhfr gene. 20 ⁇ g of total cellular DNA from A29 was mixed with 1 ⁇ g of Hind Ill-linearized pBR 322 DNA. Recipient cells were exposed to this DNA mixture and, after two weeks, methotrexate resistant colonies were picked. Genomic DNA from transformants was isolated, cleaved with Hind III and analyzed for the presence of pBR322 sequences. Two independent isolates were examined in this way and in both cases multiple copies of pBR322 sequences were present in these methotrexate transformants.
  • DNAs were obtained from masc cultures resistant to 0.1, 2,
  • the restriction maps of the mouse and hamster dhfr genes are significantly different and permit one to distinguish these genes in blot hybridization experiments. In all transformants examined, one observes two sets of restriction fragments homologous to a mouse dhfr cDNA clone: a series of bands characteristic of the endogenous mouse gene and a second series characteristic of the donor hamster gene.
  • the utility of transformation of the dhfr locus is a function of the relative frequencies both of transformation and of spontaneous resistance to mtx.
  • the demonstration that all mtx resistant L cells picked result from transformation rather than amplification of endogenous genes suggests that amplification of dhfr is a rare event in this cell line.
  • the use of a purified dhfr gene is likely to overcome these difficulties by enormously increasing the frequency of transformation.
  • the mutant dhfr gene has been used as a dominant transfer vector to introduce nonselectable genetic elements into cultured cells.
  • One experimental approach exploits the observation made previously, Wigler, M. , et al.; Cell 16: 777-785 (1979) , that competent cells integrate other physically unlinked genes at high frequency. Cultures exposed to pBR322 DNA, along with the genomic 2NA containing the mutant dhfr gene give rise to mtx resistant cell lines containing multiple copies of the bacterial plasmid.
  • An alternative approach to genetic vectoring involves ligation of pBR.322 sequences to the selectable dhfr gene prior to transformations This procedure also generates transformants containing multiple pBR322 sequences.
  • Amplification of dhfr genes results in amplification of of pBR322 sequences, but the patterns of amplification differ among cell lines. In one instance, all pBR322 sequences amplify with increasing mtx concentrations. in other lines, only a subset of the sequences amplify. In yet other lines, sequences appear to have been lost or rearranged. In some lines, amplification proceeds with increasing mtx concentrations up to 40 ⁇ g/ml, whereas in others, amplification ceases at 2 ⁇ g/ml. At present, the amplification process is not understood nor has the amplification unit been defined. Whatever the mechanisms responsible for these complex events, it is apparent that they can be expolited to control the dosage of virtually any gene introduced into cultured cells.
  • Mouse teratocarcinoma (TCC) stem cells provide a unique vector for the introduction of specific, predetermined, genetic changes into mice Mintz, B. & Illmensee, K. ,
  • TCC wt is a mouse teratocarcinoma feeder-independent cell line (6050P) with tk + (wild-type) phenotype.
  • #TCC tk- is a derivative of TCC wt that is resistant to BrdUrd and is tk-deficient.
  • #LHB 2b is a mouse L tk- cell line transformed to the tk + phenotype with the Herpes thymidine kinase gene .
  • ⁇ TCC tk-1 , -3, -4 , and -5 are HAT-resistant teratocarcinoma clones derived from TCC tk- after transformation with the Herpes thymidine kinase gene.
  • the number of viral tk gene fragments and the location of these fragments in independent transformants were examined utilizing the blot hybridization technique of Southern, Southern, E. M. , J. Mol. Biol., 98: 503-517 (1975).
  • the donor DNA was the recombinant. plasmid, ptk-1, digested to completion with Bam HI.
  • This plasmid contains a 3.4 kb fragment with the viral tk gene inserted at the single Bam HI site within the tetracycline resistance gene of pBR322. Transformation with Bam-cleaved tk DNA results in integration with loss of the Bam sites at the termini of the 3.4 kb fragment.
  • High molecular weight DNA from transformants was cleaved with Bam HI, fractionated by agarose gel electrophoresis, and transferred to nitrocellulose filters; the filters were then annealed with nick-translated 32 P-tk DNA.
  • each clone contains at least one viral tk gene.
  • each clone reveals a band of molecular weight greater than 3.4 kb. The molecular weights of the annealing fragments differ among the transformed clones, a result suggesting that integration has occurred at different sites within the DNA of the respective transformants.
  • the rate of loss per generation may then be calculated from the formula F M (1-X) N-M + F N , in which F M is the relative cloning efficiency in selective medium after M generations in non-selective medium; F N is similarly defined for N generations; and X is the rate of loss per cell generation such as TCC tk-1, were relatively stable and lost the tk + phenotype at frequencies less than 0.1% per generation in nonselective medium. Other, less stable, lines (TCC tk-2 and TCC tk-5) lost tk + expression at 2% per generation in the absence of selection.
  • Tumors were formed by inoculating syngeneic hosts (usually two hosts per clone) subcutaneously with 10 7 cells from each of the same five transformed clones. DNA from these tumors was analyzed by blot hybridization. Neutralization assays and electrophoretic mobility tests of the tk enzyme were also carried out to identify expression of the viral gene. In addition, samples of the same tumors were fixed and examined histologically for evidence of differentiation.
  • the restriction fragment profiles of the viral tk gene demonstrated that the gene was retained in all nine tumors analyzed.
  • each tumor grown without HAT selection
  • its cell line of origin cultured under HAT selective pressure
  • the number and location of the annealing fragments in seven of the tumors was identical to that of the corresponding cell line.
  • the introduced tk gene was, in most cases, maintained for many cell generations spanning at least three weeks in vivo without significant loss or translocation.
  • a gene rearrangement had occurred, resulting from the loss of the original tk-containing fragment and the appearance of a new fragment of different molecular weight. It is of interest that these two tumors were produced from the two TCC clones that lost the tk + phenotype in vitro at highest frequencies (Table II) .
  • tumors contained an array of differentiated tissues similar to those in tumors from the untransformed TCC wt and TCC tk cell lines of origin. Included were muscle, neural formations, adipose tissue, some bone, squamous keratinizing epithelium, and other epithelia, ducts, and tubules .
  • Biochemical transformants of mouse L may constitute a competent subpopulation in which an unselectable gene can be introduced, along with an unlinked selectable gene, at frequencies higher than in the general population, Wigler, M. , et al., Cell 16: 777-785 (1979).
  • Cotransformation experiments have therefore been carried out in which the Herpes viral tk gene was used as a selectable marker to introduce the human ⁇ -globin gene into tk TCC cells.
  • a cloned Hind III restriction endonuclease fragment of human chromosomal DNA containing the ⁇ -globin gene (plasmid ph ⁇ -8) was cleaved with the enzyme Hind III and mixed with Hind Ill-linearized ptk-1.
  • TCC tk- cells were exposed to these genes, they were grown for two weeks in HAT selection medium and tk + transformants were cloned and analyzed by blot hybridization fox presence of human ⁇ -globin sequences.
  • a 4.3 kb Bgl II restriction fragment containing the intact human ⁇ -globin gene is entirely contained within the donor pH -8 plasmid.
  • High molecular weight DNA from the transformants was therefore cleaved with the Bgl II enzyme and analyzed in blot hybridization using the 32 P-labeled 4.3 kb Bgl II fragment as an annealing probe.
  • the malignant stem cells of mouse teratocarcinomas have contributed a novel avenue of intervention. These cells can be grown in culture, selected for specific mutations, and microinjected into blastocysts, where they lose their neoplastic properties and participate in development, Dewey, M. , J. et al., Proc. Natl. Acad, Sci. USA, 74 : 5564-5568 (1977); Watanabe, T., et al . , Proc. Natl. Acad. Sci., 75: 5113-5117 (1978) .
  • the cultured TCC cells have therefore been viewcd as vehicles for transmitting predetermined genetic changes to mice, Mintz, B., Brook-haven Symp., Bio., 29: 82-85, (1977); Mintz, B., Differentiation 13: 25-27 (1979) .
  • Such changes obviously might include genes acquired by uptake of DNA.
  • DNA-mediated gene transfer into cells of fibroblast lines has been accomplished in culture, Wigler, M. , et al., Cell 11: 223-232 (1977); Wigler, M. , et al . , Cell 14: 725-731 (1978); Willecke, K. , et al., Molec. Gen. Genet. 170: 179-185 (1979) , Graf, L. H., et al . , Somat. Cell Genet., in press (1979); Wigler, M. , et al., Proc. Natl. Acad. Sci. USA, 76: 1373-1376 (1979) ; Wigler, M. , et al. Proc. Natl. Acad.
  • TCC-cell route for gene transfer into embryos offers the advantage that transformants, i.e., cell clones in which the specific gene has been retained, can be identified and isolated by selection or screening.
  • transformants i.e., cell clones in which the specific gene has been retained
  • cotransfer with a selectable one has been found to occur with relatively high frequency, Wigler, M. , et al., Cell 16: 777-785 (1979).
  • tk teratocarcinoma cells have been treated with the cloned thymidine kinase gene of Herpes simplex and a number of HAT-resistant tk + clones have been obtained with a frequency of about one transformant per ⁇ g of DNA.
  • mice mammary tumor virus genes, Hynes, N.E. , et al . , PNAS 78:2038-204 (1981); Buetti, E. and Diggelman ⁇ , H. , Cell 23:335-345 (1981) , remain responsive to glucocorticoids following transfer into heterologous recipients. Further, the fusion of the promoter element of MMTV to the structural gene encoding dihydrofolate reductase renders this gene inducible with glucocorticoids, Lee, F., et al. , Nature 294:228-232 (1981).
  • hGH human hormone synthesis gene introduced into the chromosome of murine fibro blasts has been examined.
  • growth hormone synthesis is restricted to the pituitary gland.
  • either glucocorticoid or thyroid hormone generates a 3-fold increase in the level of GH mRNA; when added together, a 10-fold induction is observed, Martial, J.A. , et al., PNAS 74 :1816-1820 (1977); Tusfainski, R.J. , et al., PNAS 74:2357-2361 (1977).
  • Cotransformation has been used to introduce from 1 to 20 copies of the human growth hormone (hGH) gene into thymidine kinase deficient (tk-) murine fibroblast cells which express functional glucocorticoid receptors, Lippman, M.E. and Thompson, E.B., J. Biol Chem. 249:2483-2488 (1974).
  • the administration of hormones to these cotransformed cells results in a 2.5 to 5-fold induction of hGH mRNA and a similar induction in secreted growth hormone protein in over half of these cell lines.
  • the DNA sequences responsive to induction reside within 500 nucleotides of DNA flanking the putative site of transcription initiation. Fusion of this segment of DNA to the tk structural gene renders the tk gene responsive to hormone action.
  • the human growth hormone gene is a member of a small multi-gene family composed of at least five genes, each sharing significant sequence homology and each likely to have arisen by divergence from a common ancestor, Niall, E.G., et al., PNAS 68:866-870 (1971), DeNoto, P.M. , et al. , Nucl. Acids Res. 9:3719-3730 (1981). Two of these genes have diverged significantly to generate two distinct hormones, chorionic somatomammotropin and prolactin.
  • Several different recombinant phage containing hGH sequences have been isolated from a library of human DNA constructed in the Charon phage ⁇ 4A, Maniatis, T.
  • ⁇ 20A and ⁇ 2C Two different pfaages, designated ⁇ 20A and ⁇ 2C, contain the entire growth hormone gene within a 2.6 kb Eco RI fragment (Fig. 3) .
  • the complete nucleotide sequence of this Eco RI fragment derived from ⁇ 20A indicates that this fragment contains the entire hGH gene along with 500 5' and 525 3' flanking nucleotides.
  • the restriction map of the 2.6 kb Eco RI fragment of ⁇ 2C is identical to that of ⁇ 20A.
  • Partial sequence analysis of ⁇ 2C from the 5' Eco RI site to the Bam HI site adjacent to the first exon (Fig. 1) is identical to that of ⁇ 20A. Both ⁇ 20A and ⁇ 2C, which presumably encode the major form of growth hormone, were utilized in these studies.
  • Mouse Ltk- cells express functional glucocorticoid receptor, Lippman, M.E. and Thompson, E.B., J. Biol. Chem 249:2483-248 (1974) .
  • a viral tk gene has been utilized as a selectable marker to introduce the DNA from recombinant phages ⁇ 20A and ⁇ 2C into this cell line by DNA-mediated gene transfer, Graham, F.L. and van der Eb, A.J. , Virology 52:456-467 (1973); Wigler, M. , et al., Cell 16:777-785 (1979).
  • Cotransformants were identified by blot hybridization, Southern, E.M., J. Mol. Biol.
  • DNA was isolated from nine transformnats obtained following cotransfer to tk and hGH DNA, restricted with the enzyme for Eco RI, and analyzed by blot hybridization utilizing highly radioactive hGH probes.
  • Three of the six transformants exposed to ⁇ 2C DNA integrate at least one intact Eco RI fragment containing the hGH gene. Only one of three transformants exposed to ⁇ 20A DNA contains an intact gene. The hGH copy number in these lines is variable. Some lines contain a single integrated fragment, others contain 10-20 copies of hGH DNA.
  • RNA was isolated from cells grown in the presence or absence of 10 -6 M dexamethasone for 48-72 hours.
  • Northern blot analysis of these RNA preparations reveals that two cell lines synthesize an 825 bp mRNA homologous to the hGH RNA; a 3-4 fold increase in hGH mRNA is observed upon the addition of glucocorticoid.
  • These two indicible cell lines derive from cotransformations with each of the two different clones.
  • Another cell line synthesizes an 825 bp species and a 3 kb species constitutively; no induction with dexamethasone is observed.
  • the origin of the larger transcript is unknown and may reflect either aberrant initiation, termination or splicing.
  • Growth hormone mRNA is not detectable in a fourth line. Sufficient homology exists between the mouse and human growth hormone sequences to detect the expres murine gene in these cell lines, albeit at reduced sensitivity (see below) . Tk + transformants which do not contain intact hGH sequences do not synthesize detectable GH RNA either before or after induction. Thus, the fibroblast does not synthesize appreciable levels of endogenous mouse GH mRNA.
  • hGH mRNA levels were obtained by titrating hGH sequences in RNA populations by dot blotting.
  • a standard curve was initially generated with increasing quantities of human pituitary RNA. If radioactive dots are coutned, the pituitary standard remains linear from, at least 1 to 500 pg of mRNA.
  • This assay proved highly reproducible and allows the detection of as little as 1 pg of specified hGH mRNA in 25 ⁇ g of total cellular RNA in a few hours of autoradiographic exposure.
  • the results of this analysis confirm the less quantitative characterizations obtained by Northern blotting.
  • Two clones appropriately regulate hGH RNA in response to glucocorticoid and express about 125 and 25 copies of mRNA, respectively, per cell in the fully induced state (see Table III) .
  • RNA was prepared from these transformants and screen for the presence of hGH RNA by dot blotting. Seven of nine cotransformants express hGH RNA. Poly A + RNA was then prepared from five clones and examined by Northern blot analysis. These lines synthesize an 825 bp hGH mRNA, comigrating with the human pituitary transcript, which is inducible by the addition of glucocorticoid. In several of these lines, the inducible hGH mRNA appears as a doublet; while the origin of the larger transcript is unclear, a similar doublet in rat GH mRNA in GH 3 cells is due to differential polyadenylation.
  • RNAs previously examinee for hGH sequences were subjected to Northern blot analysis and exposed to highly radioactive tk probe to test for the induction of tk gene expression in these cell lines.
  • these cotransformants were inducible for the expression of GH mRNA.
  • these cells express distinct 1.3 and 0.9 kb tk mRNA transcripts (see below) whose level remains constant before and after induction. Glucocorticoid inducibility therefore is not property of all cotransformed elements, but is specific for the hGH gene.
  • This cell line synthesizes about 15 u g per liter in the absence of hormone and 60 u g of hGH per liter in the fully induced state.
  • indue tion in the levels of hGH mRNA results in a concomitant induction in the levels of secreted protein.
  • hGH sequence element responsive to hormonal induction can impart hormone sensitivity when fused to other structural genes.
  • Expression of the 2.6 kb Eco RI fragment is hormonally regulated in mouse cells.
  • cotransformed tk gene expression is not regulated by glucocorticoids.
  • a fusion gene consisting of the 5' flanking sequences of hGH and the structural sequences encoding CK was therefore constructed to discern whether the tk gene in this configuration is responsive to glueo corticoid induction.
  • This tk fragment contains the entire structural gene but lacks all essential promoter elements, McKnight, S .L. , et al., Cell 25: 385-398 (1981).
  • a fusion gene was introduced into Ltk- cells and poly A was then isolated from resulting tk + transformants.
  • the patterns of the tk RNAs on Northern blot analysis are complex.
  • Transformants containing a wild-type tk gene frequently express two tk mRNA transcripts, 1.3 and 0.9 kb.
  • the 1.3 kb RNA is always expressed in transformants containing an intact tk gene and encodes the wild-type enzyme.
  • the 0.9 kb transcript initiates internal to the tk gene, consists solely of tk structural gene sequences, and encodes a truncated protein of diminished activity.
  • RNA from three tk + transformants containinc the hGH-tk fusion gene reveals two species of tk RNA.
  • fusion transcript 1.25 kb in length consisting of three 5' nucleotides derived from the hGH gene and the remainder encoded by the viral tk gene.
  • the size of the larger RNA in these cells is consistent with a fusion transcript.
  • the 0.9 kb RNA presumably represents the aberrant tk transcript.
  • the 0.9 kb transcript generated from an internal tk promoter provides a, fortuitous internal control.
  • the hGH-tk fusion gene generates two mRNAs; a 1.25-1.3 kb RNA presumably initiated into GH sequences and second 0.9 kb species initiating in the tk structural gene about 300 nucleotides downstream.
  • the 5' terminus of this mRNA has not been precisely defined. Only the longer RNA is hormonally inducible, suggesting that the hGH regulatory element acts locally to activate transcription and has little or no effect upon the frequency ⁇ f close, but downstream initiations. This arguments must be tempered by the fact that cell. lines expressing the fusion gene have integrated multiple copies of gene. It is therefore possible that the smaller transcript derives solely from genes which remain unresponsive to hormone action.
  • RNA when introduced into cells, synthesize significant quantities of RNA while their endogenous counterpart genes remain transcriptionally silent. Further, if appropriate control signals exist in the recipient cell, expression of the newly-introduced genes may be properly regulated.
  • Exogenous hGH genes in the murine fibroblast containing receptors express inducible levels of mRNA. Finally, expression of endogenous GH sequences in non-pituitary tissue is virtually never observed.
  • Transformed genes may therefore escape the developmental history of the cell and immediately conform to the "on" state, a state accessible to appropriate regulators. In this state, genes may be regulated if appropriate controlling elements exist within the cell . The regulated state may therefore involve "trans" acting factors such as steroid hormone-receptor complexes.
  • Ltk aprt a derivative of Ltk clone D, Kit, S. et al., Esp. Cell Res. 31:291-312 (1963) , was maintained in Dulbecco's modified Eagle ' s medium (DME) containing
  • DAP diaminopurine
  • Murine Ltk- aprt- cells are adenine phosphoribosyltrans ferase-negative derivatives of Ltk-clone D cells. Cells were maintained in growth medium and prepared for transformation as described, Wigler, M. , et al. , PNAS 76 :1373-1376 (1979).
  • HEp-2 human
  • HeLa human
  • CHO Choinese hamster ovary
  • Ltk- cells were grown in growth medium.
  • LH2b a derivative of Ltk- transformed with herpes simples virus tk DNA, was maintained in growth medium containing hypoxanthine at 15 ⁇ g/ml, aminopterin at 0.2 ⁇ g/ml, and thymidine at 5.0 ⁇ g/ml (HAT), Wigler, M. , et al.. Cell 1:223-232 (1977). All culture dishes were Nunclon (Vanguard International, Neptune, N. J.) plastic.
  • the feeder-independent mouse teratocarcinoma cell culture line 6050P Watanabe, T. , et al., PNAS 75 -.5113-5117 (1978) obtained from a tumor of the OTT 6050 transplant line, was used as the wild-type, or tk , parent and is here designated TCC wt.
  • This- line is of the X/O sex chromosome type and has a modal number of 39 chromosomes with characteristics described in Watanabe, T. , et al., (1978).
  • the cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum.
  • High molecular weight DNA was obtained from cultured cells (CHO, LH2b, and HeLa) or from frozen rabbit livers as previously described. Wigler, M. , et al.. Cell 14:725-731 (1978) . High molecular weight salmon sperm DNA was obtained from Worthington. Restriction endonuclease cleavage (Bam I, Hindlll, Kpn I, and Xba I) was performed in a buffer containing 50 mM NaCl, 10 mM Tris.HCL, 5 mM MgCl 2 , 7 mM mercaptoethanol, and bovine serum albumin at 100 ⁇ g/ml (pH 7.9). The enzyme-to-DNA ratio was at least two units/ ⁇ g of DNA, and reaction mixtures were incubated at 37°C for at least 2 hrs (one unit is the amount of enzyme that digests 1 ⁇ g of
  • nick-translated adenovirus-2 [ 32 P]DNA was incubated with 5 ul of reaction volume for at least 2 hr, cleavage products were separated by electrophoresis in 1% agarose gels, and digestion was monitored by exposing the dried gel to Cronex 2DC x-ray film.
  • HSV DNA was isolated from CV-1-infected cells as. previously described. Pellicer, A., et al., Cell 14: 133-141 (1978). DNA was digested to com letion with Kpn I (New England Biolabs) in a buffer containing 6 mM Tris (pH 7.9), 6mM MgCl 2 , mM 2-mercapto ethanol, 6 mM NaCl and 200 ⁇ g/ml bovine serum albumin.
  • the restricted DNA was fractionated by electrophoresis through 0-5% agarose gels (17 x 20 x 0.5 cm) for 24 hr at 70 V, and the 5.1 kb tk-containing fragment was extracted from the gel as described by Maxam, A. M. and Gilbert, W. PNAS 74:560- 564 (1977) and Wigler, M. , et al., Cell 14: 725-731 (1978)
  • ⁇ X174 am3 RFI DNA was purchased from Bethesda Research Laboratories. Plasmid pBR322 DNA was grown in E. coli HB 101 and purified according to the method of Clewell, D. B., J. Bacteriol. 110:667-676 (1972). The cloned rabbit ⁇ major globin gene in the ⁇ Charon 4A derivative (RSG-1) was identified and isolated as previously described. Maniatis, T., et al., Cell 15:687-701 (1978).
  • the size of the high molecular weight DNA was determined by electrophoresis in 0.3% agarose gels using herpes simplex virus DNA and its Xba I fragments as markers. Only DNA whose average size was larger than 75 kb was found to possess transforming activity in the amplification experiments.
  • plasmid DNAs were isolated from chloramphenicol amplified cultures by isopycnic centrifugation in CsCl gradients containing 300 ⁇ g/ml ethidium bromide. Transformation and Selection
  • Twice-concentrated Hepes-buffered saline (2X HBS) was prepared; it contains 280 mM NaCl, 50 mM Hepes, and 1.5 mM sodium phosphate, pH adjusted to 7.10 ⁇ 0.05.
  • DNA/CaCl 2 solution was added dropwise to an equal volume of sterile 2X HBS.
  • a 1-ml sterile plastic pipette with a cotton plug was inserted into the mixing tube containing 2X HBS, and bubbleswere introduced by blowing while the DNA was being added.
  • the calcium phosphate/DNA precipitate was allowed to form without agitation for30-45 min at room temperature.
  • the precipitate was then mixed by gentle pipetting with a plastic pipette, and 1 ml of precipitate was added per plate, directly to the 10 ml of growth medium that covered the recipient cells. After 4-hr incubation at 37oC, the medium was replaced and the cells were allowed to incubate for an additional 20 hr. At that time, selective pressure was applied. For tk + selection, medium was changed to growth medium containing HAT. For aprt + selection, cells were trypsinized and replated at lower density (about 0.5 X 10 6 cells per 10-cm dish) in medium containing 0.05 mM azaserine and 0.1 mM adenine.
  • Methotrexate-resistant transformants of Ltk- aprt- cells were obtained following transformation with 20 ⁇ g of high molecular weight DNA from A29 Mtx RIII cells and selection in DME containing 10% calf serum and 0.2 ⁇ g/ml amethopterin.
  • tk + selection cells were grown in HAT medium; for resistance to methotrexate, cells were selected in medium supplemented with 0.1 ⁇ g/ml of methotrexate. Colonies were cloned from individual dishes to assure that each transformant arose from an independent event. Ligates between A29 DNA and linearized pBR322 DNA were prepared by incubating a 1:1 ration (w/w) of Sal I-cleaved DNAs with T 4 ligase (Bethesda Research Laboratories) under the conditions recommended by the supplier.
  • a calcium phosphate precipitate was prepared using 2 ⁇ g ligate and 18 ⁇ g carrier/ml, and added to recipient cells (the amount of ligate was limited because of the observation that plasmid inhibits transformation).
  • the DNA was allowed to remain in contact with the cells for 4-12 hr and the medium was then aspirated and replaced with fresh DME. Selective pressure was applied 24 hr following exposure to DNA, After 2-3 weeks, colonies were isolated using cloning cylinders.
  • transformation was performed as described previously except that the TCC tk- cells were seeded at 3 X 10 cells/plate one day prior to transformation.
  • a calcium phosphate/DNA precipitate prepared with 4 ⁇ g of the recombinant plasmid, Ptk-1, digested with Bam H1, in the presence of 20 ⁇ g of high molecular weight DNA obtained from L tk- aprt cells.
  • some cells were treated in suspension, Willecke, K. et al., Molec. Gen. Genet. 170:179-185 (1979).
  • Ltk- aprt- mouse cells were transformed with either 1 - 10 ⁇ g of ⁇ X174, 1 ⁇ g of pBR322 or 1 ⁇ g of R ⁇ G-1 DNA in the presence of 1 ng of HSV—1 tk gene and 10-20 ⁇ g of salmon sperm carrier DNA, as previously described. Wigler, M. et al., PNAS 76:1373-1376 (1979) .
  • Tk + transformants were selected in DME containing hypoxanthine, aminopterin and thymidine (HAT) and 10% calf serum. Isolated colonies were picked using cloning cylinders and grown into mass cultures.
  • Extracts were prepared by resuspending washed cell pellets (approximately 10 7 cells) in 0.1 ml of 0.02 M potassium phosphate, pH 7, containing 0.5% Triton X-100. The supernatant (cytoplasm) obtained after 25 min of 700 X g centri fugation was used for the quantitation of enzymatic activity and for electrophoresis. aprt and protein were assayed as previously described. Chasin, L. A., Cell 2:37-41 (1974). Inclusion of 3 mM thymidine triphosphate, an inhibitor of 5 ' -nucleotidase, Murray, A. W. and Friedrichs, B. , Biochem, J.
  • the polyacrylamide gel contained an Ampholine (LKB) mixture of 0.8% pH 2.5-4, 0.8% pH 4-6, and 0.4% pH 5-7.
  • LLB Ampholine
  • cells from monolayer cultures were scraped into phosphate buffered saline and washed.
  • the cell pellet was suspended in 5 volumes of extraction buffer (0.01 M Tris.HlCl, pH 7.5, 0.01 M KCl, lmM MgCl 2 , lmM 2-mercaptoethanol, and 50 pM thymidine).
  • the cell suspension was frozen and thawed three times and the KCl concentration was then adjusted to 0.15 M.
  • the cytoplasmic extract was obtained by centrifugation at 30,000 X g for 30 min, and the supernatant was used for tk assays as described in Wigler, M. et al. Cell 16:777-785 (1979).
  • Cytoplasmic extracts from tumors were obtained after disruption of the cells in a Potter-Elvejehm homogenizer. They were then treated as described above for cultured cells.
  • One unit of thymidine kinase is defined as the amount of enzyme which converts one nanomole of thymi dine into thymidine monophosphate per minute.
  • anti-HSV-1 tk antiserum or preimmune serum was mixed with an equal volume of cytoplasmic extract, and ATP and magnesium were added to 6.7 mM.
  • the enzyme-antibody mixture was incubated for 30 min at room temperature, centrifuged at 2,000 X g for 10 rain, and the supernatant was assayed for tk activity.
  • Rabbit and mouse cDNAs were prepared by using avian myeloblastosis virus reverse transcriptase (RNA-dependent DNA polymerase) as described in Myers, J. C. and Spiegelman, S., PNAS 75:5329-5333 (1978). Isolation of Transformed Cell DNA
  • Nuclei and cytoplasm from clones ⁇ X4 and ⁇ X5 were prepared as described by Ringold, G. M, , et al. Cell 10:19-26 (1977). The nuclear fraction was further fractionated into high and low molecular weight DNA as described by Hirt, B. , J. Mol. Biol. 26:365-369 (1967).
  • Cellular DNA was digested with restriction endonucleases, electrophoresed on agarose slab gels, transferred to nitrocellulose filter sheets, and hybridized with 32 P-labeled DNA probes as described by Wigler, M. et al. , PNAS 76:1373-1376 (1979).
  • DNA from transformed cells was digested with various restriction endonucleases using the conditions specified by the supplier (New Engi-nd Biolabs or Bethesda Research Laboratories). Digestions were performed at an enzyme to DNA ratio of 1.5 U/ ⁇ g for 2 hr at 37°C. Reactions were terminated by the addition of EDTA, and the product was electrophoresed on horizontal agarose slab gels in 36 mM Tris, 30 mM NaH 2 PO 4 , 1 mM EDTA (pH 7.7). DNA fragments were transferred to nitrocellulose sheets, hybridized and washed as previously described. Weinstock, R. , et al., PNAS 75: 1299-1303 (1978) with two modifications. Two nitrocellulose filters were used during transfer.
  • RNA was electrophoresed through 1% agarose slab gels (17 X20 X 0.4 cm) containing 5 mM methylmercury hydroxide as described by Bailey, J. and Davidson, N. , Anal. Biochem. 70:75-85 (1976). The concentration of RNA in each slot was 0.5 ⁇ g/yl. Electrophoresis was at 110 V for 12 hr at room temperature.
  • RNA was transferred from the gel to diazotized cellulose paper as described by Alwine, J. C. , et al. , PNAS 74 :5350- 5354 (1979) by using pH 4.0 citrate transfer buffer. After transfer, the RNA filter was incubated for 1 hr with transfer buffer containing carrier RNA at 500 ⁇ g/ml. The RNA on the filters was hybridized with cloned DNA probe at 50 ng/ml. labeled by 32 P-nick translation, Weinstock, R., et al. , PNAS 75:1299-1303 (1978) to specific activities of 2-8 X
  • reaction volumes were 25 pl/cm 2 of filter.
  • Hybridization was in 4X standard saline citrate (0.15 M
  • filters were soaked in two changes of 2X standard saline citrate/25 mM sodium phosphate/1.5 mM sodium pyrophosphate/0.1% sodium dodecyl sulfate/5 mM EDTA at 37°C for 30 min with, shaking to remove formamide. Successive washes were at 68°C with 1X and 0.1X standard saline citrate containing 5 mM EDTA and 0.1% sodium dodecyl sulfate for 30 min each.
  • Murine Ltk cells were maintained in Dulbecoo's modified Eagle's medium (DME) supplemented with 10% calf serum (M.A. Bioproducts). Tk + transformants were selcted and maintained in DME, 10% calf serum, 15 ⁇ g/ml hypoxanthine, 1 ⁇ g/ml aminopterin, 5 ⁇ g/ml thymidine (HAT) , Wigler, M. , et al., Cell 11:223-232 (1977).
  • DME Dulbecoo's modified Eagle's medium
  • HAT thymidine
  • Ltk- cells were transformed with 1 ng ptk and 1 yg of either hgH ⁇ clone DNAs or hGH plasmid DNA per 10 cells, as described previously, Wigler, M. , et al., Cell 16:777-785 (1979). In transformations with GH-tk fusion gene, 5-10 ng of the plasmid was used per 10 cells; transformation efficiency of this construct is approximately 10-fold lower than ptk.
  • hGH mRNA in L cells was quantitated by dot blotting, Thomas, P., PNAS 77:5201-5205 (1980) using a minifold filtration manifold (Schleicher and Schuell).
  • Samples contained a total of 20 g RNA (specific RNA plus carr ier ribosomal RNA) , in 2 X SSC in a total volume of 25 ⁇ L, and were loaded without vacuum.
  • the filters were soaked at least 5 hours in 20 X SSC. After loading, vacuum was applied and individual wells were rinsed 3 X with 6 X SSC. Filters were then baked and hybridized exactly as for Northern blots. Dots were visualized by exposure to X-ray film and signals quantitated by punching out dots and liquid scintillation counting.

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