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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• This invention concerns the introduction and expression of genetic informational material, i.e., DNA which includes genes coding for proteinaceous materials and/or genes regulating or otherwise influencing the production thereof, into eucaryotic cells, that is, cells of organisms classified under the Superkingdom Eucaryotes including organisms of the Plant and Animal Kingdoms.
• 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.
• eucaryotic cells specifically mammalian cells, were transformed with foreign DNA coding for a selectable phenotype.
• This work has been extended and has resulted in the present invention wherein 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.
• it has unexpectedly been discovered that such foreign DNA can be expressed by the cotransformants to generate functional proteins.
• 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.
• the present invention provides major advances over bacterial systems for future use in the commercial prepar- ation 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 for such proteinaceous materials. After cellular synthesis, the precursor can be further processed or converted within the eucaryotic cell to produce the desired molecules of biological significance. This phenomenon is well known for insulin which is initially produced in the eucaryoti cell as preproinsulin which is then converted to active insulin within the cell by appropriate peptide cleavage.
• 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 the production of preproinsulin, not insulin.
• insulin a relatively small and well characterized protein, this difficulty can be overcome by chemical synthesis of the appropriate gene, such an approach is inherently limited by the level of understanding of the amino acid sequence of the desired protein.
• clotting factors, antibodies and uncharacterized enzymes for which the exact amino acid sequence is not yet known, a procaryotic system will likely not prove satisfactory.
• a eucaryotic system is not associated with such disadvantages since the eucaryotic cell possesses the necessary processing machinery It is thus one important object of the present invention to provide a process for producing desired proteinaceous materials such as interferon protein, insulin, antibodies and the like which does not require a detailed molecular understanding of amino acid sequence.
• interferon is a glycoprotein containing sugar molecules in addition to protein. If produced in a bacterial cell, the interferon lacks the sugar molecules which are added when interferon is produced in a human cell.
• proteinaceous materials produced within bacteria may include endotoxins which can cause inflammation if the proteinaceous material is administered to a mammal without significant purification. By contrast, interferon produced in a eucaryotic cell would be free of endotoxins.
• the present invention provides a process for inserting a foreign DNA I to which an inducible promoter DNA III sequence has been linked into a eucaryotic cell which involves cotransforming the cell with this combination or hybrid of foreign DNA I and DNA III and with unlinked DNA coding for a selectable phenotype not otherwise expressed the eucaryotic cell.
• This cotransformation is carried out in suitable medium and in the presence of selective conditions permitting survival or identification of eucaryotic cells which have acquired the selectable phenotype.
• This invention also provides processes for producing proteinaceous and other materials comprising cotransforming. eucaryotic cells as described hereinabove and maintaining the cotransformed cells under suitable conditions including the presence of an agent capable of inducing promoter DNA III to repeatedly transcribe DNA I to form mRNAs and translate the mRNAs to form protein or ultimately other products.
• cotransformation with a foreign DNA I to which a promoter DNA III has been joined 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 in a suitable medium and in the presence of an antagonist permitting survival or ident fication of eucaryotic cells which have acquired the dominant selectable phenotype and in the presence of an inducing agent for promoter DNA III.
• this invention provides methods for the therapeutic treatment of genetically defective eucaryotic cells and for the treatment of patients suffering from genetically-based illnesses or diseases.
• FIG. 1 is a schematic flow diagram illustrating the cotransformation process.
• FIG. 2 is a schematic flow diagram illustrating a process for recovering foreign DNA I from cotransformed cultured cells using double selection techniques.
• 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.
• Phenotype 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 are secreted by the cell and can be detected as new phenotypes either by functional, immunologic or biochemical assays.
• Interferon protein means the proteinaceous part of the glycoprotein interferon, that is, the portion remaining after removal of the sugar portion. It includes the protein portion of interferon derived from human leukocy 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.
• foreign DNA I can be inserted into any eucaryotic cell by cotrans forming the cell with DNA I and with unlinked foreign DNA II which incl udes a gene coding for a s electable phenotype not expressed by the cell unl ess acquired by transformation .
• the cotransformation is carried out in a suitable growth medium and in the presence of s elective conditions such that the only cells which survive or are otherwise al tered are those which have required the selectable ph enotype . 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 proteinaceous materials, the genes for which may be inserted into and expressed by eucaryotic cells using the cotransformation process include interferon protein, insulin, growth hormones, clotting factors, viral antigens, enzymes and antibodies.
• DNA I and DNA II may not need be purified to obtain integration and expression, it is oftentimes preferable that the DNAs be purified prior to use in cotransforming cells. Such purification limits the possibility of spurious results due to the presence of contaminants and increases the probability that cotransformed cells can be identified and stably cultured. Also, although not essential, it is sometimes desirable that 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 eucaryotic chromosomal DNA. Additionally, it is preferable, that DNA I and DNA II be treated with calcium phosphate prior to use in cotransforming eucaryotic cells.
• the procedure for so treating DNA with calcium phosphate is set forth more fully hereinafter Finally, 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 I and/or DNA II are attached to phage DNA and then encapsidated in phage particles prior to cotransforma tion.
• any DNA II coding for a selectable phenotype would be useful in the cotransformation 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 transferase.
• 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 cotransformation involves DNA I which is physically and chemically unlinked to DNA II, and the DNA I is stably integrated into the chromosomal DNA within the nucleus of the cotransformed 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 techniques well known in the art. Additionally, the cells can be 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 biopolymer for which synthetic genes are constructed.
• the instant invention provides a process for producing novel proteins not yet in existence. Additionally, it provides a process for producing proteins which, although they presently exist, do so in such minute quantities or in such impure form that their isolation and/or identification cannot otherwise be effected. Finally, the invention provides a process for producing partially proteinaceous products such as the glycoproteins 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 inserting a multiplicity of foreign DNA I molecules into a eucaryotic cell comprises cotransforming th.e 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 medium and in the presence of an agent permitting survival and/or identification of cells 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 (DNA II) survive and/or are identified. These cells then also contain multiple copies of DNA I.
• This approach is particularly appropriate for the insertion of multiple copies of amplifiable genes which confer drug resistance upon the cell, e.g., the mutant dihydrofolate reductase gene which renders cells resistant to methotrexate.
• Cotransformed eucaryotic cells which have. acquired multiple copies of DNA I may then be used to produce increased amounts of the gene product for which 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 molarity basis, of an agent which permits survival and/or identification 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 molarity basis
• genes associated with drug resistance e.g., the gene for dihydrofolate reductase which renders cells resistant to methotrexate, are particularly suitable.
• multiple 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.
• 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 g-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 heterologous 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 Hin Ill-cleaved chicken DNA wit Hin Ill-cleaved plasmid pBR 322 results in the formation of hybrid DNA molecules in which the aprt gene is now adjacent to plasmid sequences. Transformation of aprt cells is now performed with this DNA. Transformants should contain the aprt gene covalently 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 ligas
• One such fragment should contain the aprt gene covalently linked to pBR 322 sequences coding for an origin of replication and the ampicillin resistant marker. 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. More recently, a series of cotransformation experiments 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 the human ⁇ globin gene. A series of experiments is now under way to determine whether this human gene is expressed after induction in these murine cells.
• heterolo gous human globin genes can be expressed in this system, a series of exceedingly interesting experiments can be per formed utilizing iii vitro constructed mutants along with natural mutants of genes derived from thalassemi ⁇ indivviduals to determine: a) the role of flanking 5 r and 3 r sequences in the induction process; b) the significance of a linked gene arrangement observed in this locus in controlling expression; and c) the metabolic nature of the defect in the variety of ⁇ and & thalassemic states.
• 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 if linked with the promoter sequence. Cotransformed cells may then produce markedly elevated levels of the protein for which the gene codes in the presence of DMSO.
• the promoter sequence joined to a desired gene might be used in cotransformation with an amplifiable gene for drug resistance. They might be used with a desired gene such as one for an antibody to cotransform eucaryotic cells.
• 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 or other material
• Another illustration of a possible inducible promoter se- quence is human growth hormone gene.
• the human growth hormone gene in pituitary cells is tightly controlled by levels of thyroid hormone and corticosteroid.
• Rat pituitary cells, GH-3 synthesize increasing amounts of rat growth hormone in response to the addition of corticosteroids or thyroid hormone. Therefore, attempts will be made to introduce the cloned human growth hormone gene into these cells and determine whether this heterologous gene is placed under hormonal control in its new cellular environment. If this can be effected, one can then begin to alter this gene, creating a series of deletions of DNA from the 5 1 and 3 1 termini of the transcript to discern whether hormonal induction is maintained in these deletion mutants. In this way, one may localize any potential sequences which may render a gene responsive to hormone action.
• this system may provide a promoter which is inducible in the presence of hormone and can be used in cotransforma tion, with or without amplification, in the manner described hereinabove.
• cotransformation may provide an approach to genetic therapy. Specifically, it may be possible to therapeutically treat and/or cure genetically defective eucaryotic cells in order to alleviate symptoms associated therewith by cotransforming the defective cells with a gene for a selectable marker and with a genetically correct gene.
• the genetically-based defective cells may then be cotransformed with a competent globin gene and with a gene conferring drug resistance as a marker, the cotransformation being carried out in the presence of the drug.
• These cells can be transplanted back into the patient.
• the patient is then 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. Ultimately, this approach will 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 experi ments 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 ⁇ g of Pst-cleaved ⁇ X replicative form DNA. This DNA was then added to mouse Ltk cells using the transformation conditions described under Methods and Materials hereinafter. After 2 weeks in selective medium (HAT), tk transforma 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.
• HAT selective medium
• ⁇ X sequences in transformed cells were determined by subcellular fra ⁇ tionation. 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.
• 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 woul d 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 s major globin gene which was isolated from a cloned library of rabbit chromosomal DNA (Maniatis , T . , et al . , Cell 15 : 687- 701
• R 6G-1 conis ts of a 15 kb rabbit DNA fragment carried on the bacteriophage cloning vector Charon 4a .
• Intact DNA from this clone (R6 G-1 ) was mixed with the viral tk DNA at a molar ratio of 100 : 1 , and tk trans formants were isol ated and examined for the presence of rabbit globin sequences .
• Cl eavage 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 nicktranslated to generate a probe for subsequent anneal ing experiments .
• the ⁇ -globin genes of mouse and rabbit are partially homologous , although we do not observe annealing of the rabbit ⁇ -globin probe with Kpn-cl eaved mouse DNA under our experimental conditions .
• cleavage of rabbit liver DNA with Kpn I generates the expected 4 . 7 kb globin band .
• Cleavage of trans formed 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.
• 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 variable.
• 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 s electable marker .
• This cotransformation sys tem should allow the introduction and stable integration of virtually any defined gene into cul tured cells . Ligation to either viral vectors or selectable biochemical markers is not required.
• At least one of the rabbit ⁇ -globin mouse transformants expresses polyadenylated 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.
• Hybridization analysis of restriction endonuclease-cleave 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.
• Cotransformants contain at least one copy of the tk 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 rabbit ⁇ -globin gene provide an opportunity to study the expression and subsequent processing 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 trans formation 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 culture.
• the number of rabbit globin genes integrated in thes e 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 . Tnese 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 heterologous 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. Because homology exists between the mouse and rabbit globin sequences, it was necessary to determine experimental conditions such that the rabbit globin cDNAs did not form stable hybrids with mouse globin mRNA but did react completely with homologous rabbit sequences.
• 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 6 protected
• RNA from this transformant was fractionated into nuclear and cytoplasmic populations to determine the intracellular localization of the rabbit globin RNA.
• the cytoplasmic RNA was further fractionated by oligo (dT) -cellulose chroma tography into poly (A) + and poly (A) - RNA .
• Poly (A) + cytoplasmic RNA from clone 6 hybridizes with the rabbit cDNA with an
• R 0 t 1 ⁇ 2 of 25 ⁇ his value is 1/80 th of the R 0 t1 ⁇ 2 observed with total cellular RNA, consistent with the observation that poly (A) + cytoplasmic RNA is 1-2% of the total RNA in a mouse cell . Hybridization is not detectable with either nuclear RNA or cytoplasmic poly
• 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 % locali zed 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 electrophqresed on a methyl-mercury/agarose gel . Bailey , J . & Davidson, N. , Anal . Biochem. 70: 75-85 (1976) , and transferred to diazotized cellulose paper.
• RNA on the filters was hybridized with'DN ⁇ from the plasmid p ⁇ Gl , which contains rabbit g-globin. cDNA sequences . Maniatis , T. , et al . . Cell 8 : 163-182 (1976) .
• a discrete 9S species of RNA was observed in the cytoplasm of the transformant, which comigrated with rabbit globin mRNA isolated from rabbit erythroblasts .
• 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 .
• the hybridization of mature rabbit mRNA to RBG1 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 cf 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. Hybridization with a specific 5' probe showed that the internal 222 base pair fragment was present.
• 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 alterna tives.
• globin sequences in transformed fibroblasts can be expressed 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 gene is repressed by factors that are partially overcome by a gene dosage effect provided by the introduction of 20 additional globin genes. Finally, 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 net 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 ⁇ -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 cyto plasmic 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. et al., Nature 265: 687-695 (1977).
• Purified tk gene 500 pg was mixed with 1-10 ⁇ g 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) , tk transformants were observed at a frequency of one colony per 10 cells per 20 pg of purified gene. Clones were picked and grown into mass culture.
• 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-l One ⁇ -globin clone, designated R G-l 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 molar ratio of 100:1, and tk transformants were isolated and examined for the presence of rabbit globin sequences. Cleavage of RSG-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 expressed 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 70oC, 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 th 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 x1776.
• 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 phenotype. Although the frequency of transformation in nature is not known, this process could have profound physiologic and evolutionary consequences.
• 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 wild- type 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 gen thus rendering these cells insensitive to elevated levels 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, 32 P-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 mass cultures resistant to 0.1, 2, 10 and 40 ⁇ g/ml methotrexate, and the copy number of pBR322 and dhfr sequences was determined by blot hybridization. Six independent transformed lines were examined in this fashion. Five of these lines exhibited multiple bands homologous to pBR322 sequences. In four of these transformed clones, at least one of the pBR 322-specific bands increased in intensity upon amplification of dhfr. In SS-1, two pBR322-specific bands are observed in DNA from cells resistant to 0. l ⁇ g/ml methotrexate. These bands increase several-fold in intensity in cells resistant to 2 ⁇ g/ml.
• mutant dhfr gene can be used as vector for the introduction and amplification of defined DNA sequences into cultured animal cells.
• 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 DNA containing the mutant dhfr gene give rise to mtx resistan cell lines containing multiple copies of the bacterial plasmid.
• 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 resis tant to BrdUrd and is tk-def icient.
• #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 Hl site within the tetracyclin 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.
• Clones were picked and giown in HAT selective medium for 40 cell generations . Cells were then grown in nonselective medium for 28 or 150 generations prior to determining their cloning efficiencies under selective and nonselective conditions . One hundred cells were plated in triplicate into HAT selective and nonselective media . The relative cloning efficiency in selective medium is defined as the ratio of the cloning efficiency under selective conditions to the cloning efficiency under nonselective conditions ( 50- 70 %) . #In these calculations it is assumed that for any given cell line the rate of loss of the tk phenotype is cons tant in each cell genera tion .
• 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-sel ective medium; F N is similarly defined for N generations ; and X is the rate of loss per cell generation.
• F M is the relative cloning efficiency in selective medium after M generations in non-sel ective medium
• F N is similarly defined for N generations
• X is the rate of loss per cell generation.
• TCC tk-1 were relatively stable and lost the tk + phenotype at frequencies less than 0.1% per generation in nonselective medium.
• 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. In two instances, however, 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 los t the tk + phenotype in vitro at highest frequencies (Table II) .
• tumors contained an array of differentiated tissues s imilar to those in tumors from the un trans formed 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 selectahle 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 After 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 hybridi zation for 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 B ⁇ l II enzyme and analyzed in blot hybridization using the 32 P-labeled
• 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 viewed 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. Sci., in press (1980); Lewis, W. H.
• TCC-cell route for gene transfer into embryos offers the advantage that transformants , i . e . , cell clones in wh ich the specific gene has been retained , can be identified and isolated by selection or screening .
• transformants i . e .
• cotransfer with a selectable one has been found to occur with relatively high frequency , Wigler, M. , et al . , Cell 16 : 777- 785 (19 79 ) .
• 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.
• globin gene Many genes of interest in a developmental context are not selectable.
• An example is the globin gene.
• a fragment of human genomic DNA containing an intact ⁇ -globin gene was administered to TCC tk- cells along with the unlinked HSV tk gene. This proved to be an effective method to obtain TCC tk + clones m which, from hybridization evidence, the human ⁇ -globin gene was present.
• 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 DME.
• DME Dulbecco's modified Eagle's medium
• 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 contain ⁇ ing 50 mM NaCl, 10 mM TrisoHCL, 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 ⁇ l 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-, 6 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 (RBG-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 bubbleswer.e 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 transforma tion).
• 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 5 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 HI, 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 yg 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 cells) in 0.1 ml of 0.02 M potassium phosphate, pH 7, containing 0.5% Triton X-100.
• the super natant (cytoplasm) obtained after 25 min of 700 X g centri f ⁇ ation 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
• 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
• 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 enzvme 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 min, and the supernatant was assayed for tk activity.
• Rabbit and mouse cDNAs were prepared by using avian myeloblastosis virus reverse transcriptase (RNA- ⁇ epencent DNA polymerase) as described in Myers, J. C. and Spiegelman, S.,
• 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 DN 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 England Biolabs or Bethesda Research Laboratories) . Digestions were performed at an enzyme to DNA ratio of 1.5 U/ ⁇ g for 2 hr at 37oC. 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/ ⁇ l. 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 ⁇ l/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 68oC with 1X and 0.1x standard saline citrate containing 5 mM EDTA and 0.1% sodium dodecyl sulfate for 30 min each.

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