JPH09500783A - Bifunctional selective fusion gene based on cytosine deaminase (CD) gene - Google Patents

Abstract

(57) Summary The present invention provides a selective fusion gene comprising a dominant positive selection gene in reading frame with a negative selection gene and fused to a negative selection gene. The selection fusion gene encodes a single bifunctional fusion protein capable of conferring a dominant positive and negative selection phenotype on the host cell. The dominant negative selection phenotype is conferred by the cytosine deaminase (CD) gene for 5-fluorocytosine sensitivity (5-FC ^s ). A dominant positive selection phenotype is conferred by, for example, the neo gene for G-418 aminoglycoside antibiotic resistance (G-418 ^r ) or the hph gene for hygromycin B resistance (Hm ^r ). The present invention further provides a recombinant expression vector, such as a retroviral vector, containing a selective fusion gene, and a cell into which the recombinant expression vector has been introduced. The selectable fusion genes of the present invention are expressed and regulated as a single genetic material, allowing for highly efficient co-regulation and co-expression.

Description

Detailed Description of the Invention           Based on the cytosine deaminase (CD) gene                     Bifunctional selective fusion genebackground   The present invention broadly relates to genes that express a selective phenotype. More specifically, the book The invention is capable of expressing both a dominant positive selection phenotype and a negative selection phenotype. Related genes.   Genes that express a selection phenotype can be identified and expressed in host cells into which the gene has been introduced. It is widely used in recombinant DNA technology as a means to separate. Typically Genes that express a selective phenotype are expressed in host cells as part of a recombinant expression vector. Will be introduced. A positive selection gene keeps the introduced gene in a stable form. It provides a means for identifying and / or isolating possessing cells. And This ability allows these genes to facilitate gene transfer and analysis of gene function. I do. On the other hand, the negative selection gene is a cell that retains the introduced gene. Provide a means to eliminate.   Many genes are available that confer a selective phenotype on animal cells. Bacteria Neomycin phosphotransferase gene (neo) (Colbere-Garapin et al., J. . Mol. Biol. 150: 1,1981), hygromycin phosphotransferase inheritance Hph (Santerre et al., Gene 30: 147, 1984), and xanthine-guaninephos. Horibosyl transferase (gpt) gene (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78: 2072, 1981), Used as the dominant positive selection gene. Herpes simplex virus type I Thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11: 223, 1977); cell origin Native adenine phosphoribosyl transferase (APRT) gene (Wigler et al., Proc . Natl. Acad. Sci. USA 76: 1373, 1979); and hypoxanthine phosphoribosi. Letransferase (HPRT) gene (Jolly et al., Proc. Natl. Acad. Sci. USA 80 : 477, 1983) is commonly used as a recessive positive selection gene. Outline Thus, dominant selection genes are more versatile than recessive genes. Because it is recessive While gene use is limited to mutant cells that are defective in selectable function, , Because the dominant gene can be used in wild-type cells.   Some genes confer negative and positive selection phenotypes. I can. These include the HSV-I TK, HPRT, APRT, and gpt genes. This These genes drive the conversion of nucleoside or purine analogs into cytotoxic intermediates. It encodes an enzyme that catalyzes. The nucleoside analogue ganciclovir (ganci clovir; GCV) is an effective substrate for HSV-I TK, but not for cell-derived TK. It is a sufficient substrate. Therefore, this is relative to the HSV-I TK gene in wild type cells. Can be used for all negative selections (St. Clair et al., Antimicrob. Agents Chemot her. 31: 844, 1987). However, the HSV-I TK gene has cell-derived TK activity. To the missing mutant cells However, it can only be used effectively for positive selection. Similarly, the above HPRT and The use of the APRT gene for either positive or negative selection is HPRT respectively^-Cell or APRT^-Limited to cells (Fenwick, "HGPRT System", 33 Pp. 3-373, M.S. Gottesman (ed.), Molecular Cell Genetics, John Wiley and Son S., New York, 1985; Taylor et al., "APRT System", pages 311-332, M.S. Gottesman ( Ed.), Molecular Cell Genetics, John Wiley and Sons, New York, 1985). one On the other hand, the gpt gene is positively and negatively selected in wild type cells. Can be used for any of Negative to gpt gene in wild-type cells Selection is possible with 6-thioxanthine. This is due to the bacterial gpt enzyme , A cell-derived HPRT enzyme that is effectively converted to a cytotoxic nucleotide analog (Besnard et al., Mol. Cell. Biol. 7: 4139, 1987).   Another negative selection gene was recently described by Mullen et al. (Proc. Natl. Acad. Sci. USA). 89:33, 1992). The bacterial cytosine deaminase (CD) gene , 5-Fluorocytosine (5-FC) is converted to 5-Fluorouracil (5-FU). 5-FU is In addition, intracellularly, 5-fluoro-uridine-5'-triphosphate and 5-fluoro- It is metabolized to 2'-deoxy-uridine-5'-monophosphate. These are RNA and And inhibits DNA synthesis and causes cell death. Therefore, 5-FC carries the CD gene The expressing cells can be effectively eliminated. CD gene is found in normal cells And not positively selectable.   More recently, a genetic design designed for use in human gene therapy. There has been interest in selection genes that can be incorporated into the offspring transfer vector. gene Therapy may, for example, alter cell activity or cell phenotype, or alternatively By introducing a heterologous gene that can express the drug required for treatment, It can be used as a means to enhance cell function. Gene therapy also includes one or more It can be used for the treatment of congenital genetic diseases caused by defects or deletions of a number of genes. These diseases collectively lead to significant morbidity and mortality. Examples of such genetic diseases Include hemophilia A and B (deficiency of blood coagulation factors VIII and IX, respectively). Caused by), α₁Antitrypsin deficiency and adenosine Aminase deficiency is included. In each of these particular cases, Gene has been identified and its complementary DNA (cDNA) has been cloned as a molecule. (Wood et al., Nature 312: 330, 1984; Anson et al., Nature 315: 683, 1984; And Long et al., Biochemistry 23: 4828, 1984; Daddona et al., J. Chem. Biol. Chem. 259: 12 101, 1984). Palliative therapy is applied for some of these genetic disorders (usually , In the form of blood product administration or transfusion), while one of the treatments for such genetic diseases The method is to introduce a replacement for the missing or missing gene into the somatic cells of the patient. is there. This process is called "gene therapy" (Anderson, Science 226: 40). 1, 1984).   The process of gene therapy typically involves (1) taking somatic (non-germ) cells from the patient. (2) Ex vivo treatment or replacement Introducing the gene into cells via a suitable vector capable of expressing therapeutic or alternative genes And (3) transplant or transfuse these cells into the original patient A process (wherein the therapeutic or alternative gene is expressed and has some therapeutic benefit) Including). Somatic gene transfer for human gene therapy is currently being investigated. Achieved in Sivo (Kasid et al., Proc. Natl. Acad. Sci. USA 87: 473, 1990. Rosenberg et al., N .; Engl. J. Med. 323: 570, 1990), and this relatively efficient The bad process is that the cells that have the alternate gene introduced (before returning them to the patient) Facilitated by the use of a dominant positive selection gene to determine and isolate Will be. For example, the neo gene is used in human gene therapy It has been used to identify genetically modified cells.   However, in some cases the introduction of genetically modified cells does May endanger the health of the patient. In vivo (i The ability to selectively remove (in vivo) allows the procedure to be reversed. This gives patients an additional margin of safety for gene therapy. this That means negative selection (that can function in wild-type cells) (ie, “suicide ") Can be achieved by incorporating the gene into a vector. Dominant positive Choice Incorporation of genes capable of conferring a phenotype and a negative selection phenotype is the Co-expression and co-regulation (co-expression) of alternative and negative selection phenotypes -regulation) and can minimize the size of the vector. However Et al., Positive selection for the gpt gene in some cases measures Precise selection conditions that can be difficult are required. The reason is that the gpt gene Dominant positive selection phenotype and negative selection rather than using Co-expression with the phenotype is a function of two different genes (these are the other dominant positives). And separately encoding negatively selectable functions) It is generally achieved.   Dominant positive selection phenotype and negative encoded by different genes Existing strategies for co-expressing the active selection phenotype are usually complex. Give a difficult problem. The most widely used technique is to code the two phenotypes separately. Co-transfecting the two plasmids Wigler et al., Cell 16: 777, 1979). However, the efficiency of co-transfer Seldom becomes 100%, and the two genes independently It may undergo developmental regulation. The second strategy is to combine two genes into a single gene. Two independent transcription units were ligated on the lasmid or placed in the viral vector. It is to arrange as a unit. This method also regulates these genes independently Done It has a disadvantage of getting it. In the case of retrovirus vector, on the other hand Suppression of the other independent transcription unit can occur (Emerman and And Temin, Mol. Cell. Biol. 6: 792, 1986). In addition, many functional promotions Despite the successful construction of a retrovirus vector carrying Under certain circumstances, the virus vector contains two functional transcription units. There may not be enough space to obtain (Overell et al., Mol. Cell. Biol. 8:18. 03, 1988). The third strategy is to combine two genes into one 2cistronic mRNA. Then, it expresses using a single promoter. However, this According to the method, the distal open reading frame is often inconsistent Translated efficiently (and usually reduced) in Kaufman et al., EMBOJ. 6: 187, 1987. ), And how such an expression strategy is effective in primary cells. Not at all.   The present invention provides a dominant positive selection phenotype encoded by different genes. And a negative selection phenotype more effectively and reliably provide.                             Summary of the Invention   The present invention is in reading frame with a negative selection gene and Selective fusion inheritance containing a dominant positive selection gene fused to a gative selection gene Providing a child (selectable fusion gene). This selection fusion gene has a single bifunctional It encodes a fusion protein. This protein has a dominant positivity for host cells. A negative selection phenotype and a negative selection phenotype can be provided. The selective fusion gene of the present invention is , Nucleoses from the bacterial cytosine deaminase (CD) gene for negative selection It contains a tide sequence.   In a preferred embodiment, the selected fusion gene is a nucleo from the neo gene. It contains a nucleotide sequence from the bacterial CD gene fused to the tide sequence. This genetic The offspring are referred to herein as the CD-neo selection fusion gene (SEQ ID NO: 1). this The CD-neo selection fusion gene was used for G-418 resistance (G-418^r)When 5-Fluorocytosine sensitivity for negative selection (5-FC^s) And give.   The present invention also provides a recombinant expression vector (eg, a recombinant expression vector) containing the selected fusion gene. Rovirus), and cells into which the recombinant expression vector has been introduced.   Since the selective fusion gene of the present invention is expressed and regulated as a single gene body, , Enables highly efficient co-regulation and co-expression.                         Brief description of the drawings   FIG. 1 shows the plasmids tgCMV / hygro / LTR, tgCMV / neo, tgCMV / hygro-CD, tgCMV / CD. -illustrates the expression cassettes contained in hygro, tgCMV / neo-CD and tgCMV / CD-neo . Horizontal arrows indicate the transcription start site and transcription direction. Labeled as LTR Was Open frames are retroviral long terminal repeats. Outlines labeled CMV The frame is the cytomegalovirus promoter.   FIG. 2 was prepared from a pool of transfected NIH / 3T3 cells. The result of the cytosine deaminase assay regarding an extract is shown. This extract is It was assayed by measuring the conversion of tocin to uracil.   FIG. 3 shows the retrovirus vector tgLS (+) neo and the used in the present invention. Figure 2 shows a schematic representation of the proviral structure of TgLS (+) CD-neo.   Figure 4 shows non-infected NIH / 3T3 (lane 1), tgLS (+) neo-infected NIH / 3T3 (lane 2), And tgLS (+) CD-neo-infected NIH / 3T3 (lane 3) cell pool The result of Nase assay is shown. This result shows that cells infected with tgLS (+) CD-neo It is shown to express high levels of cytosine deaminase activity.   Figure 5 shows non-infected NIH / 3T3 cells (plates a, b and c) and tgLS (+) neo ( Plates d and e) or tgLS (+) CD-neo (plates f and g) retrovirus 5 shows a photograph of a stained colony of NIH / 3T3 cells infected with A. niger. Cells grow long term In the assay, medium alone (plate a) or G-418 (plates b, d and f) or G-418 + 5-FC (plates c, e and g) are grown in medium supplemented Was done. Data show that uninfected NIH / 3T3 cells are sensitive to G-418 and resistant to 5-FC. And NIH / 3T3 cells infected with tgLS (+) neo of G-418 and 5-FC NIH / 3T3 cells resistant to both and tgLS (+) CD-neo resistant to G-418 , And was sensitive to 5-FC.                         Detailed description of the invention   SEQ ID NO: 1 and SEQ ID NO: 2 (shown immediately before the claims) are the CD of the present invention. -neo The specific sequence of the nucleotide sequence of the fusion gene and the corresponding amino acid sequence. An embodiment is shown. The CD-neo selection fusion gene shown in the sequence listing is the neo gene (nucleo CD gene (nucleotides 4 to 1281) linked to a sequence derived from tide 1282 to 2073 ) Derived sequence.Definition   As used herein, the term "selection fusion gene" refers to negative selection Fused to an alternative gene and in reading frame with a negative selection gene A nucleotide sequence containing a dominant positive selection gene at It confers a dominant positive and negative selection phenotype on the cellular host. Encoding a single possible bifunctional fusion protein. "Dominant positive selection inheritance "Progeny" encodes a protein that confers a dominant positive selection phenotype on the cellular host. Nucleotide sequence, and is discussed and exemplified in more detail below. It is. A "negative selection gene" is one that confers a negative selection phenotype on the cell host. Protein Described nucleotide sequence and is also discussed and discussed in more detail below. And exemplify. "Selection gene" generally refers to a dominant positive selection gene and Gattive selection gene.   Expression product of the selection gene (ie, the protein encoded by the selection gene) Are fused by peptide bonds and the biological activity of each of the two proteins A selection gene, if at least a portion of the "Fused to and in reading frame with another selection gene." For the CD-neo selection fusion gene disclosed herein, the CD protein and ne o Proteins are fused by peptide bonds, and a single bifunctional fusion tamper When expressed as a protein, the CD gene (encoding cytosine deaminase -Fluorocytosine sensitive, ie 5-FC^sGives a negative selection phenotype of , Neo gene (encoding neomycin phosphotransferase, G-418 resistance, Ie G-418^rGive a dominant positive selection phenotype), and neo relic It is in the reading frame with the gene.   The selection gene sequences of the components of the invention are preferably contiguous; The component selection gene sequence is an internal non-translated nucleotide sequence such as an intron. It is possible to construct a selective fusion gene that is divided by. Of the object of the present invention In order to express the selected fusion gene, the expression product of the selected A single two fused by a tide bond Such a non-adjacent selection gene sequence will be fused if a functional fusion protein is produced. It is considered to be combined.   A "nucleotide sequence" is a deoxyribonucleoside, such as a DNA or RNA sequence. Refers to a heteropolymer of tide or ribonucleotide. Nucleotide sequence Are either in the form of separate fragments or as a component of a larger construct. Exists. Preferably, the nucleotide sequence is prepared according to standard biochemical methods, eg For example, cloning vectors can be used to identify, manipulate, and recover sequences Is the amount or concentration. Recombinant nucleotide sequences can be cloned, Products of various combinations of digestion and ligation steps. This process is Construction with structural coding sequences distinguishable from homologous sequences found in natural systems Produces things. Generally, a structural coding sequence, eg, a selection fusion gene of the invention, is coded for. The nucleotide sequences to be added include nucleotide fragments and short oligonucleotides. Assembled from an otide linker, or assembled from a series of oligonucleotides, It may provide a synthetic gene that can be expressed in the alternative transcription unit. Such an array is , Preferably an internal untranslated sequence typically present in eukaryotic genes, ie Presented in the form of an open reading frame uninterrupted by the intron Provided. The genomic DNA containing the relevant selection gene sequence is preferably a selection gene. Used to obtain the appropriate nucleotide sequence encoding Lagment may also be used. like this If the sequence does not interfere with the manipulation or expression of the coding region, the untranslated DNA sequence is May be 5'or 3'from the open reading frame, or It may be in the open reading frame. However, some genes Introns that are necessary for proper expression in the host. For example, HPRT election Alternative genes contain introns that are required for expression in embryonic stem (ES) cells. the above As suggested by, the nucleotide sequences of the invention may also include RNA sequences. An example For example, where the nucleotide sequence is a retrovirus for infecting a cellular host. Packaged as RNA in the virus. The use of retrovirus expression vector , Discussed in more detail below.   The term "recombinant expression vector" refers to transfection or viral infection. Can be transduced into target cells and encode the expression of a selection fusion gene A replicable unit of a form of DNA or RNA. This selective fusion gene is One or more genetic elements that have a regulatory role in gene expression, eg, transcription. Control of transcription initiation sequence and transcription termination sequence and translation initiation sequence and translation termination sequence Below, it is transcribed into mRNA and translated into protein. The recombinant expression vector of the present invention Are replicated in bacterial cells and, for example, calcium phosphate precipitates. Directly into target cells by starch, electroporation or other physical transfer methods It can take the form of a DNA construct to be transfected. Takes the form of an RNA construct Recombinant expression vectors can be pretranslated using, for example, a proviral DNA vector. Produce retroviruses that are defective and contain RNA transcripts of proviral DNA. Infectious retrovirus packaged by the appropriate "packaging" cell line It can be in the form of a loose. The host cells are infected with the retrovirus and Torovirus RNA is stably integrated into the chromosomal DNA of the host cell to produce the provirus Replicated by reverse transcription into a double-stranded DNA intermediate that forms Then, Purowill DNA is expressed in host cells to produce the polypeptide encoded by the DNA. To achieve. Therefore, the recombinant expression vector of the present invention is present in infectious retroviruses. Stable integration into chromosomal DNA in appropriate host cells as well as existing RNA constructs Reverse transcript of the isolated retroviral RNA genome, or a cloned copy thereof. Contains a copy of the proviral DNA or retroviral DNA It also includes a cloned copy of the missing intermediate form. Proviral DNA is a provirus When the sequence is expressed in an infected host cell, it is transcribed into mRNA and Transcription independently operably associated with selected structural DNA sequences translated into protein Including factors. The recombinant expression vector used for direct transfection is It contains a DNA sequence that enables the vector to replicate in a bacterial host cell. In the present invention Various recombinant expression vectors suitable for use in the following are described below.   The term "(introduce)" means that the recombinant gene containing the selected fusion gene is It means the introduction of the current vector into cells. The transduction method is physical in nature Get (ie, transfection), that is, the method of transcribing into RNA Packaged in infectious viral particles and infect target cells DNA used to deliver (ie, infect) the desired genetic material Relying on the use of recombinant viral vectors (such as retroviruses) . Many different types of mammalian gene transfer and recombinant expression vectors developed (See, for example, Miller and Calos, Gene Transfer Vectors for Mammal. lan Cells ”Current Comm. Mol. Biol., (Cold Spring Harbor Laboratory, New  York, 1987)). Naked DNA transferred to calcium phosphate (Berman et al., Proc. Natl. Acad. Sci. USA 84 81: 7176, 1984), DEAE-De. Kistran transfection (McCutchan et al., J. Natl. Cancer Inst. 41: 351. , 1986; Luthman et al., Nucl. Acids Res. 11: 1295, 1983), protoplast fusion (D eans et al., Proc. Natl. Acad. Sci. USA 84 81: 1292, 1984), electroporation (Potter et al., Proc. Natl. Acad. Sci. USA 84 81: 7161, 1984), Lipofec. (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987), Polybrene. Hexadimethrin bromide transfection omide transfection) (Kawai and Nishizawa, Mol. Cell. Biol. 4: 1172, 1984) Gene Transfer by Laser Micropuncture of Cell and Cell Membrane (Tao et al., Proc. Natl. . Acad. Sci. U SA 84: 4180, 1987), including (but not limited to) many techniques. One of them is physically introduced into a mammalian cell by transfection. Can be Development of various infection techniques utilizing recombinant infectious virus particles for gene delivery Has been issued. This represents the preferred approach to the present invention. Smell this way The viral vector that has been used is Simian virus 40 (SV40; Karlsson et al. , Proc. Natl. Acad. Sci. USA 84 82: 158, 1985), adenovirus (Karlsson et al. , EMBOJ. 5: 2377, 1986), adeno-associated virus (LaFace et al., Virology 162: 483, 1). 988) and retrovirus (Coffin, 1985, pp. 17-71, edited by Weiss et al., RNA Tumor Vi. ruses, 2nd edition Vol. 2, Cold Spring Harbor Laboratory, New York) Includes loose vectors. Therefore, there are many gene transfer and expression methods. However, it is essential to introduce and express genetic material in mammalian cells. Function. Some of the above techniques transduce hematopoietic or lymphoid cells These have been used for the calcium phosphate transfection (Berman et al., Supra, 1984), protoplast fusion (Deans et al., Supra, 1984), et al. Lectroporation (Cann et al., Oncogene 3: 123, 1988), as well as recombinant adeno Virus (Karlsson et al., Supra; Reuther et al., Mol. Cell. Biol. 6: 123, 1986), Deno-associated virus (LaFace et al., Supra) and retroviral vector (Overell et al., Oncogene 4: 1425, 1989). Primary T lymphocytes are elect Population (Cann et al., Supra, 1988) and retroviral infection (Nishihara et al., Cancer Res. 48). : 4730, 1988; Kasid et al., Supra, 1990).Construction of selective fusion gene   The selective fusion gene of the present invention has a dominant positivity fused with a negative selection gene. It contains a selection gene. Selection genes are commonly expressed, for example, in antibiotic resistance expression. Molds, or can supply autotrophic requirements (for dominant positive selection), Preferably encodes a protein that can activate toxic metabolites (for negative selection) It includes a gene that does. The DNA sequence encoding the bifunctional fusion protein has a recombinant Constructed using DNA technology, dominant positive selective genes and negative Individual DNA fragments encoding the selection gene are assembled into an appropriate expression vector. It's The 3'end of one selection gene is linked to the 5'end of another selection gene, The reading frame for sequences within a system allows mRNA sequences to Allows translation into a bifunctional fusion protein. Single selection fusion gene Expressed under the control of the promoter.   A dominant positive selection gene is a stable trait when transduced into a host cell. It is a gene that expresses a dominant phenotype that allows positive selection of transfectants. Departure The bright dominant positive selection gene preferably encodes resistance to the antibiotic G418. Tn5-derived aminoglycoside phosphotransferase Ze gene (neo or aph) (Colbere-Garapin et al., J. Mol. Biol. 150: 1, 1981; Sou thern and Berg, J. Mol. Appl. Genet. 1: 327, 1982); and Hygromai Syn resistance (Hm^r) Hygromycin-B phosphotransferr conferring a selective phenotype of Gene (hph or "hygro") (Santerre et al., Gene 30: 147, 1984; Sugden et al., Mol. Cell. Biol. 5: 410, 1985; ATCC accession number 39820, plasmid pHEBol Available from). Hygromycin B interferes with transposition Aminoglycoside inhibitors that inhibit protein synthesis by inducing mistranslation It is a raw material. The hph gene phosphorylates the antibiotic hygromycin B and is nontoxic Of the Hph gene into Hm^rgive. Other acceptable Potential dominant positive selection genes include: Neomycin phosphotran Bacterial neo gene encoding spherase (Beck et al., Gene 19: 327, 1982); Mikoff Xanthine-guanine phospho from Escherichia coli encoding resistance to enolic acid Ribosyl transferase gene (gpt) (Mulligan and Berg, Proc. Natl. Ac ad. Sci. USA 78: 2072, 1981); a necessary and increased level of purine biosynthesis. To select cells constitutively expressing Bell DHFR, the drug methotrexate (MTX Dihydrofolate-derived from murine cells or E. coli that can be competitively inhibited by Cutase (DHFR) gene (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495, 1983; Simonsen et al., Nucl. Acids Res. 16: 2235, 1988); typhimuri um histidinol Dehydrogenase (hisD) gene (Hartman et al., Proc. Natl. Acad. Sci. USA 85:80. 47, 1988); E. coli tryptophan synthase β subunit (trpB) gene (Hart man et al., supra); puromycin-N-acetyltransferase (pac) gene ( Vara et al., Nucl. Acids Res. 14: 4117, 1986); Adenosine deaminase (ADA) inheritance Offspring (Daddona et al., J. Biol. Chem. 259: 12101, 1984); multiple drug resistance (MDR) gene (K ane et al., Gene 84: 439, 1989); mouse ornithine decarboxylase (OCD) gene (Gupba and Coffino, J. Biol. Chem. 160: 2941, 1985); E. coli asparagine Acid transcarbamylase catalytic subunit (pyrB) gene (Ruiz and Wahl, Mol . Cell. Biol. 6: 3050, 1986); and encodes asparagine synthetase E. coli asnA gene (Cartier et al., Mol. Cell. Biol. 7: 1623, 1987).   The negative selection gene provides stable transduction when transduced into host cells. A gene that expresses a phenotype that allows negative selection (i.e. elimination) of the body You. The preferred negative selection gene of the present invention confers 5-fluorocytosine sensitivity. Bacterial CD Gene Encoding Cytosine Deaminase (Genbank Accession No. X63656) It is.   Other enzymes suitable for negative selection are phosphate-containing prodrugs (etopo Cid-phosphate, doxorubicin-phosphate, mitomycin phosphate Alkaline phosphatase useful in the conversion of toxic dephosphorylation metabolites; For converting sulfate-containing prodrugs to free drugs Useful arylsulfatases; peptide-containing prodrugs as free drugs Proteases useful for converting, such as Serratia proteases, thermolytics , Subtilisin, carboxypeptidase and cathepsin (cathepsin B and And L); D-amino acids useful for converting prodrugs containing D-amino acid substituents. Lanyl carboxypeptidase; glycosylated prodrug as free drug Carbohydrate cleaving enzymes useful for converting (β-galactosidase and neuraminidase , Etc.); to convert a drug induced using β-lactam into a free drug -Lactamase useful for; and phenoxyacetyl group or phenylacetyl Each of these groups is used as a free drug for a drug derived at the nitrogen atom. A penicillin amidase useful for converting (penicillin V amidase or penicillin Phospho-G amidase and the like), but is not limited thereto.   Another enzyme prodrug combination is para-N-bis (2-chloroethyl) amino. Bacterial (eg, from Pseudomonas) enzymes with benzoylglutamic acid Includes Cypeptidase G2. Cleavage of the glutamate moiety from this compound is a poison Releases benzoic acid mustard. Penicillin-V amidase is doxorubicin And the phenoxyacetamide derivative of melphalan to toxic metabolites.   Due to the degeneracy of the genetic code, nucleotide sequences encoding the same amino acid sequence There can be considerable variation in the rows; examples The illustrative DNA embodiment corresponds to the nucleotide sequence in SEQ ID NO: 1 of the Sequence Listing. It is an embodiment of Such variants include one or more substitutions, deletions, Or has a modified DNA or modified amino acid sequence with additions and the net effect is Retain biological activity, and such variants are disclosed herein Substitutions can be made for specific sequences. Sequences of selected fusion genes including CD and neo Contains all or part of the CD and neo sequences, and is suitable for moderately stringent conditions (50 Hybridizes to the nucleotide sequence of SEQ ID NO: 1 in the sequence listing under 2 × SSC) Is possible and expresses a biologically active fusion protein, They are equivalent.   A "biologically active" fusion protein is a selective phenotype of a constituent selection gene. Amino acid sequence similarities sufficient to provide the amino acid sequence similarity of the invention disclosed herein. Share with specific embodiments.   In a preferred embodiment, sequences from the bacterial cytosine deaminase (CD) gene Fused with sequences from the bacterial neomycin phosphotransferase (neo) gene Is done. The obtained selection fusion gene (called CD-neo selection fusion gene) is G-418.^r And 5-GC^sAnd have a single dominant positive and negative selection phenotype. Encodes a bifunctional fusion protein that can be expressed and regulated as a genetic entity . The CD-neo selective fusion gene is used in a patient population likely to receive ganciclovir. Can be particularly advantageous.Recombinant expression vector   The selective fusion gene of the present invention identifies the host cell into which this selective fusion gene is introduced. , Isolated, or removed. This selective fusion gene is By transducing a recombinant expression vector containing this selective fusion gene into It is introduced into the main cells. Such host cells include mammalian cells or insect cells. Cell types derived from higher eukaryotic origin, such as, or lower prokaryotic origin Cell types.   As mentioned above, such a selection fusion gene is preferably a gene of this selection fusion gene. Components of a recombinant expression vector capable of expressing offspring intracellularly and conferring a selective phenotype And then introduced into specific cells. Such recombinant expression vectors generally include If it contains a selection fusion gene operably linked to appropriate transcriptional or translational control sequences. Synthetic or native nucleotide sequence, eg, origin of replication, any for regulated transcription An operator sequence, a suitable promoter linked to the gene to be expressed and Enhancers, and other 5'or 3'flanking nontranscribed sequences, and 5'or 3'untranslated sequences (e.g., necessary ribosome binding sites, polyadenylation sites, sp Lysing donor and acceptor sites), and transcription termination sequences are included. You. Such regulatory sequences may include mammalian, viral, microbial, or insect gene Can be derived from The nucleotide sequence is When they are functionally related to each other, they are operably linked. For example, A lomomotor is a selective fusion gene if it controls transcription of the selective fusion gene. Operably linked to the offspring; or, the ribosome binding site is a fusion gene of choice. Is translated into a single bifunctional fusion protein , Operably linked to a selective fusion gene. Generally, operably linked is , Which means that they are adjacent.   Specific recombinant expression vectors for use in mammalian, bacterial, and yeast cell hosts -Pouwels et al., (Cloning Vectors: A Laboratory Manual, Elsevier, New Yo rk, 1985) and are well known in the art. Set used for animal cells For a detailed description of recombinant expression vectors, see Rigby, J. et al. Gen. Virol. 64: 255, 1983); Eld er et al., Ann. Rev. Genet. 15: 295, 1981; and Subramani et al., Anal. Biochem. 1 35: 1.1983. Suitable recombinant expression vectors are also viral vectors. -, In particular retroviruses (described in detail below).   The selection fusion gene of the present invention is preferably a strong enhancer and promoter. -Placed under the transcriptional control of the expression cassette. Examples of such expression cassettes include Tomatomegalovirus immediate early (HCMV-IE) promoter (Boshart et al., Cell 41: 521 , 1985), β-actin promoter (Gunning et al., Proc. Natl. Acad. Sci. USA). 84: 5831, 1987), histone H4 promoter (Guild et al., J. Virol. 62: 3795, 1988). , Mouse metallothionei Promoter (Mclvor et al., Mol. Cell. Biol. 7: 838, 1987), rat growth hormone. Promoter (Miller et al., Mol. Cell Biol. 5: 431, 1985), human adenosine Aminase promoter (Hantzapoulos et al., Proc. Natl. Acad. Sci. USA 86: 351. 9, 1989), HSV TK promoter (Tabin et al., Mol. Cell. Biol. 2: 426, 1982), α- 1 Antitrypsin enhancer (Peng et al., Proc. Natl. Acad. Sci. USA 85: 8146 , 1988), and immunoglobulin enhancers / promoters (Blankenstein et al., Nucleic Acid Res. 16: 10939, 1988), SV40 early or late promoter, Ade Two major late promoters of novirus, or polyoma virus, From sipapilloma virus, or other retrovirus or adenovirus Included are other viral promoters that have been induced. Immunoglobulin (Ig) inheritance Offspring promoters and enhancer elements have significant specificity for B lymphocytes. (Banerji et al., Cell 33: 729, 1983; Gillies et al., Cell 33: 717, 1983; Mas on et al., Cell 41: 479, 1985), these elements were found to be associated with β-glob only in red blood cells. Controls transcription of phosphogene function (van Assendelft et al., Cell 56: 969, 1989). Lap Using known restriction and ligation techniques, suitable transcription control sequences can be Intact DNA, to be excised from a DNA source and expressed according to the invention. It can be integrated in operable relationship with a selection fusion gene. Therefore, many Transcription control sequences have been successfully used in retroviral vectors to Inserted heredity Offspring can be expressed.Retro virus   Retroviruses are highly efficient in introducing the selective fusion gene of the present invention into eukaryotic cells. Can be used to transduce the Preferred for delivering offspring. In addition, retroviral integration is controlled. In a different manner, resulting in one or several copies of new genetic information per cell. Stable installation.   Retroviruses are a class of viruses whose genome is in RNA form. Retroviral genomic RNA encodes viral proteins that include: Includes trans-acting gene sequences that interact with RNA in the nucleus of viral particles Structural proteins that are linked (encoded by the gag region); make DNA complements (po reverse transcriptase; encoded by the 1 region; and particle lipoprotein envelope And bind the virus to the host cell surface during infection (env region). Envelope glycoprotein (encoded by region). Replication of retroviruses Cis-acting elements, such as proviral DNA and necessary for viral replication It is regulated by a promoter for transcription of other nucleotide sequences. Made in Sith The element used is about 600 base pairs present at the 5'and 3'ends of the retroviral genome. Within or next to two identical untranslated long terminal repeats (LTRs) of Touching. Retrovirus RNA-directed DNA polymerase or reverse transcription Primers can be used to copy their RNA genomes into double-stranded DNA intermediates by reverse transcription. To duplicate. The DNA intermediate is within the chromosomal DNA of an avian or mammalian host cell. Incorporated into. The integrated retroviral DNA is called the provirus. Provirus is a template for RNA strand synthesis for the formation of infectious viral particles. Function as. Forward transcription of provirus and assembly into infectious viral particles (a ssembly) has an endogenous trans-acting gene required for viral replication Occurs in the presence of the appropriate helper virus.   The retrovirus is in the endogenous trans of the proviral form of the retrovirus. One or more of the genes that act may be recombinant therapeutic genes, or in the context of the present invention. In this case, the selected fusion gene is replaced, and then the recombinant provirus is transformed into the cell. By introducing it, it is used as a vector. For genes that act on trans Contains the gag, pol, and env genes, which are A protein, enzyme reverse transcriptase, and envelope protein components Code (constituents). These are all required for the production of undamaged virions You. A recombinant retro deletion of the gag, pol, and env genes that act in trans The virus is unable to synthesize proteins that are essential for replication, and therefore replication-defective (rep lication-defective). Such replication-defective recombinant retroviruses are Are grown using a packaging cell line. these Packaging cell line acts on all trans necessary for the production of undamaged virions It contains an integrated retroviral genome, which provides the gene sequence for like this Proviral DNA sequences transduced into different packaging cell lines are transcribed into RNA. Infectious virus that is duplicated and contains a selective fusion gene (and / or therapeutic gene) Gag, p, which is a gene product that is encapsidated in the on but acts in trans lacks ol and env and encapsidates RNA within particles to infect other cells Unable to synthesize the gag, pol, and env proteins required for. So got The infectious retroviral vector that is capable of infecting other cells, The gene may be incorporated into the cellular DNA of the host cell. Mann et al. (Cell 33: 153, 1983) For example, to produce a stock of recombinant retrovirus without helper virus To develop various packaging cell lines (eg, Ψ2) that can be used to It is described. Ecotropic virus envelope (eg , Ψ2) -encoding capsi in a cell line having a trans-acting element Decoupling provides ecotropic (limited host range) progeny virus . Alternatively, a cell line containing an amphotropic packaging gene (eg, PA317, ATCC CRL 9078; Miller and Buttimore, Mol. Cell. Biol. 6: 2895, 1986) The assembly provides an amphotropic (wide host range) progeny virus.   Many proviral constructs have been used to express foreign genes Used successfully (eg Coffin, Weiss et al. (Eds.), RNA Tumor Virus, Second Edition, Volume 2, (Cold Spring Harbor Laboratory, New York, 1985, pp.17-71) See). Most proviral elements are derived from murine retroviruses. However, retroviruses applicable for use in accordance with the present invention include any avian or sire. Derived from a mammalian cell source. A suitable retrovirus is a retrovirus vector. The cells that should be the receptors for the new genetic material to be transduced Must be dyeable. Examples of suitable retroviruses include avian retroviruses. (For example, avian erythroblastosis virus (AEV), avian leukemia virus (ALV), avian myeloblast) Cystosis virus (AMV), avian sarcoma virus (ASV), Fujinami sarcoma virus (FuSV), spleen Visceral necrosis virus (SNV), and Rous sarcoma virus (RSV); bovine leukemia virus (B LV); cat leukemia virus (FeLV) or feline sarcoma virus (FeSV) Torovirus; murine retrovirus such as murine leukemia virus (MuLV); Us mammary tumor virus (MMTV), and murine sarcoma virus (MSV); and humans T cell lymphocyte virus 1 and 2 (HTLV-1 and -2), and ape sarcoma virus Primate retroviruses such as SSV are included. Many other suitable retro Viruses are known to those of skill in the art. For classification of retroviruses, see Weiss et al. Ed.), RNA tumor virus, 2nd edition, Volume 2, (Cold Spring Harbor Laboratory, N ew York, 1985, pp.1-160) by Teich. Combined with the present invention Good to use The retroviruses are the Moronei murine leukemia virus (MoMLV) and the Moronei murine. Sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMSV), and Kirsten It is a murine retrovirus known as murine sarcoma virus (KiSV). Mo The sequence required to construct a retrovirus vector derived from the MSV genome is pMV (A Can be obtained in combination with a pBR322 plasmid sequence such as TCC37190), but K-BAL The cell line producer of KiSV in B cells was deposited as ATCC CCL 163.3. RSV L PRSV derived from pBR322 containing TR and intact neomycin drug resistance marker Neo deposits are available from ATCC under Deposit No. 37198. A plastic containing the SNV genome Smid pPB101 is available as ATCC 45012. The retrovirus virus Rus genomes integrate their viral genomes into the chromosomal DNA of infected host cells. However, once integrated, the cells into which the infectious virus is introduced are functionally Other proviral elements that code for viral proteins that act in active trans Replication-defective Used to construct retroviral vectors.   The selective fusion gene of the present invention transduced by a retrovirus is a retrovirus. Selected under the transcriptional control of enhancers and promoters that are integrated in the LTR of R. Heterologous transcription control sequences that place alternative fusion genes or insert them between LTRs It is expressed by being put under the control of. Heterologous transcription control sequence and LTR transcription control sequence The use of both columns makes the therapeutic residue in the vector Allows for co-expression of the gene and selection fusion genes, and thus the desired therapeutic gene. It allows the selection of cells which express the specific vector sequence encoding the product. High The therapeutic gene and / or the selection fusion gene are Transcription of strong heterologous enhancer and promoter expression cassettes in the virus It may need to be under control. Many different heterologous enhancers and promoters Have been used to express genes in retroviral vectors. You. Such enhancers or promoters include those that include the mammalian genome. It may be derived from an Ils source or a cell source, and is preferably native in nature. It is constitutive. Such a heterologous transcription control sequence is a recombinant expression vector. As described above. The above gene transfer method so that it is expressed in transduced cells The DNA sequence introduced by either of the Is expressed under the control of   Particularly preferred recombinant expression vectors include pLXSN, pLNCX, and pLNL6, and Include those derivatives. These are Miller and Rosman, Biotechnique s 7: 980, 1989. These vectors retrovirally import heterologous DNA. Can be expressed under the transcriptional control of the LTR or CMV promoter, and the neo gene It can be expressed under the control of the early region promoter or the retroviral LTR. The present invention The neo gene is selected for bifunctionality as disclosed herein for use in It is replaced with a fusion gene, for example the CD-neo selection fusion gene. useful Construction of successful replication-defective retroviruses is a matter of routine skill. The resulting recombinant layer Torovirus can be integrated into the chromosomal DNA of infected host cells, but once integrated When introduced, the cells into which the infectious virus is introduced act on the functionally active trans. , Without other proviral inserts encoding viral proteins that It cannot undergo replication to produce infectious virus.Use of bifunctional selective fusion genes   The selective fusion gene of the present invention is a somatic cell (somatic cell) into which this selective fusion gene is introduced.  cells) as a means to identify, isolate, or remove cells such as Is particularly preferred for use. In gene therapy, somatic cells are removed from the patient And a recombinant expression vector containing the therapeutic gene and the selected fusion gene of the invention. It is introduced and then reintroduced into the patient. Used as a vehicle for gene therapy Obtained somatic cells include hematopoietic (bone marrow-derived) cells, keratinocytes, hepatocytes, endothelial cells, and And fibroblasts are included (Friedman, Science 244: 1275, 1989). Alternatively, Gene therapy uses injectable vectors to transduce somatic cells in vivo Can be achieved by The potential for gene transfer in humans has been demonstrated (K asid et al., Proc. Natl. Acad. Sci. USA 87: 473, 1990); Rosenberg et al. Engl. J. Med. 323: 570, 1990).   The selective fusion gene of the present invention is genetically modified in vivo. It is particularly useful for removing viable cells. Of cells expressing a negative selection phenotype In vivo ablation has been used in gene therapy as a means of ablating cell grafts. Is particularly useful in, and thus provides a means of reversing gene therapy procedures. For example, the anti-herpesvirus drug ganciclovir is used as an immunoglobulin promoter. When administered to transgenic animals expressing the HSV-I TK gene from It has been shown that selective excision of cells expressing the TK gene occurs (Heyman et al. , Proc. Natl. Acad. Sci. USA 86: 2698, 1989). Same transgenic mau GCV also showed complete regression of abelson leukemia virus-induced lymphoma (regr ession) has been shown (Moolten et al., Human Gene Therapy 1: 125, 199). 0). In a third study, murine sarcoma (K3T3) became a retrovirus expressing HSV-I TK. They were infected and transplanted into syngeneic mice. Tumors induced by sarcoma cells Completely eradicated after treatment with GCV (Moolten and Wells, J. Natl. Cancer In st. 82: 297, 1990).   The selective fusion genes of the present invention are also described in Oldfield et al., Human Gene Therapy 4: 39,199. Useful in the treatment of tumor resection, as performed by 3. tgLS (＋) CD-neo Packaging cells (about 10⁶~Ten⁹) Is the tumor Inoculated intra-tumorally. A few days later (in the meantime, the newly produced retro Virus infects rapidly growing tumor cells in close proximity), (tgLS (+) CD-neo Torovirus vector Patients receive approximately 50-200 mg of 5-FC / kg of body weight daily (orally or Or via vein) and selectively remove the infected tumor cells.   The bifunctional selective fusion gene of the present invention also promotes gene modification by homologous recombination. It can be used to proceed. Reid et al., Proc. Natl. Acad. Sci. USA 87: 4299, 1990 recently described homologous recombination in ES cells using the HPRT gene. A two-step procedure for genetic modification by "uto" homologous recombination) has been described. State briefly And this procedure involves two steps: the "in" step, here the HPRT gene. Pups embedded in the target gene sequence and transfected into HPRT host cells, The homologous recombinants in which the HPRT gene was incorporated at the target position in HAT medium They are identified by their proliferation and genomic analysis using PCR. The second "out" In the process, the desired replacement sequence embedded within the target gene sequence is included (but HP The construct (without the RT gene) was transfected into cells and the replacement sequence HPRT containing homologous recombinants (but not HPRT gene)⁺Negative against cells Isolate by active selection. This procedure introduces a selection gene sequence into the target gene Introducing a few mutations into the target gene without^-For cell lines Requires positive selection of transformants. Because the HPRT gene is positive This is because it is recessive with respect to the choice of the active. Furthermore, in ES cells Large 9-kbp HPRT minigene for inefficient expression of the HPRT gene Must be used. This gene allows for the construction and propagation of homologous recombination vectors. Let them work together. The selective fusion gene of the present invention provides an improved means by which More "in-out" homologous recombination may be performed. The selective fusion gene of the present invention is Any wild type cell can be used as it has a preponderance for positive selection. That is, it is not limited to the use of cells defective in selective phenotype). In addition, selection fusion The size of the vector containing the combined gene is significantly reduced compared to the large HPRT minigene Is done.   By way of illustration, the CD-neo selective fusion gene is used as follows: In the “in” step, the CD-neo selective fusion gene was embedded in the target gene sequence and The cells are transfected and the CD-neo selective fusion gene is The integrated homologous recombinants were grown in medium containing G-418 and then PCR was used. It is identified by performing a genome analysis. Then CD-neo⁺Second out of the cell Used in the process. Where it contains the desired replacement sequence embedded in the target gene sequence Transfect the construct (without the CD-neo selective fusion gene) into cells You. CD-neo using 5-FC⁺Selective removal of cells and subsequent genomic analysis using PCR To isolate homologous recombinants.                               Example                              Example 1        CD-neo selective fusion gene containing plasmid vector                        Build and characterize   A.Construction of bifunctional CD-neo selective fusion gene   The plasmid tgCMV / hygro / LTR (Fig. 1) contains the following factors: HCMV IE94 promoter (Bos Balt-SstII fragment containing hart et al., Cell 41: 521, 1985); mammalian cells Consensus translation initiation sequence (GCCGCCACCATG) (Kozak et al., Nucl. Acids Res . 15: 8125, 1987) containing an oligonucleotide conforming to the sequence; The hph gene encoding synphosphotransferase (Kaster et al., Nucl. Acids Res. 11: 6895, 1983) from nucleotide 234-1256; containing a polyadenylation sequence MoMLV (Shinnick et al., Nature 293: 543, 1981) from nucleotide 7764 to the 3'LTR. At pML2d containing the bacterial origin of replication (Lusky and Botchan, Nature 293) : 79, 1981) -derived NruI-AlwNI fragment; β-lactamase gene-containing p It consists of the AlwNI-AatII fragment from GEM1 (Promega Corp.).   Plasmids tgCMV / neo, tgCMV / CD, tgCMV / CD-hygro, tgCMV / neo-CD, and tgC MV / CD-neo are all structurally similar to tgCMV / hygro / LTR, and consensus translation Contains the starting sequence, but each contains a different sequence instead of the hph sequence. Plastic Sumido tgCMV / neo is an oligonucleoside that encodes three amino acids (GGA TCG GCC). Neomycin phosphotransferase instead of the tide and hph sequences (Beck et al., Gene 19: 327, 1982) containing nucleotides 154-945 derived from the bacterial neo gene. Have. The plasmid tgCMV / CD replaces the hph sequence with cytosine deaminase. Nucleotides 1645-292 from the encoding bacterial CD gene (Genbank approval number X63656) Contains 5. The CD sequence was determined by PCR using the plasmid pCD2 (Mullen et al., Proc. Natl. Aca. d. Sci. USA 89:33, 1992). The plasmid tgCMV / hygro-CD is used for hph From the hph gene, fused to nucleotides 1645-2925 from the CD gene instead of the column Containing nucleotides 234-1205. The plasmid tgCMV / CD-hygro contains the hph sequence Instead, it was fused to nucleotides 234-1256 from the hph gene and cloned from the CD gene. Contains Cleotide 1645-2922. The plasmid tgCMV / neo-CD replaces the hph sequence And an oligonucleotide encoding three more amino acids (GGA TCG GCC), From the bacterial neo gene fused to nucleotides 1645-2925 from the CD and CD genes Contains Leotide 154-942. The plasmid tgCMV / CD-neo contains, instead of the hph sequence, Nucleotide 16 from the CD gene fused to nucleotides 154-945 from the neo gene Contains 45-2922.   Standard methods such as (Ausubel et al., Current Protocols in Molecular Biology  (Wiley, New York), 1987) was used to construct the plasmid tgCMV / hygro / LTR: First tgLS (＋) HyTK (Lupton et al., Mol. Cell. Biol. 11: 3374, 1991) derived from the long chain HindIII-StuI fragment. The HCMV IE94 promoter from tgLS (-) CMV / HyTK (Lupton et al., Supra, 1991). Spanning HindIII-StuI fragment and a fragment containing the human IL-2 cDNA sequence. Plasmid HyTK-CMV-IL2 was constructed by ligating with the This human IL -2 fragments containing the cDNA sequence 5'-CCCGCTAGCCGCCACCATGTACAGGATGCAACTCC-3 'and The human IL-2 cDNA was included by PCR using 5'-CCCGTCGACTTAATTATCAAGTCAGTGTT-3 '. Amplified from After amplification, the PCR product is first treated with T4 DNA polymerase. Treatment to make the ends blunt, and the fragments from tgLS (+) HyTK and tgLS (-) CMV / HyTK Prior to ligation, it was then cut with NheI. Construction of plasmid tgCMV / hygro / LTR Therefore, the SV40 polyadenylation signal of tgCMV / hygro (Lupton et al., Supra, 1991) The SalI-PvuI fragment spanning the Moloney leukemia virus LTR from HyTK-CMV-IL2 SalI-PvuI containing (containing the retroviral polyadenylation signal) Replaced with ragment.   Plasmid tgCMV / n was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. Constructed eo: PvuI-NheI fragment spanning HCMV IE94 promoter from tgCMV / hygro. The NheI-HindIII fragment extending to the neo gene from tgLS (+) neo (Hin The dIII site was treated with T4 DNA polymerase to blunt the ends) and H Moronei leukemia virus derived from yTK-CMV-IL2 SalI-PvuI containing the LTR (containing the retroviral polyadenylation signal) The fragment was ligated.   Plasmid tgCMV / C was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. Constructed D: PvuI-NheI flag spanning HCMV IE94 promoter from tgCMV / hygro The synthetic DNA fragment (which is an oligonucleotide 5'-CTAGCCGCCACCATGTCGAATAACGCTTTACAAACAATTATTAACGCCCG-3 'and Anneal 5'-GTAACCGGGCGTTAATAATTGTTTGTAAAGCGTTATTCGACATGGTGGCGG-3 ' , PCD2 (Mullen et al., Proc. Natl. Acad. Sci. USA 89: 33, 1992) containing the remainder of the CD coding region from BstE2-AluI fragment. , And Moronei leukemia virus LTR derived from HyTK-CMV-IL2 (retrovirus poly Ligated to the SalI-PvuI fragment containing the adenylation signal). Prior to ligation, the SalI site in the latter fragment was treated with T4 DNA polymerase to terminate the ends. Was smoothed.   Plasmid tgCMV / C was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. D-hygro was constructed: a long chain ClaI-SalI fragment derived from tgCMV / CD was used as an oligonucleotide. Is leotide 5'-CCCATCGATTACAAACGTAAAAAGCCTGAACTCACCGCGAC-3 'and Amplified from tgCMV / hygro by PCR using 5'-GCCATGTAGTGTATTGACCGATTCC-3 ' ClaI-NcoI fragment (the PCR product was cut with ClaI and NcoI before ligation), and And tgCMV / hygro / LTR It was ligated to the NcoI-SalI fragment containing the rest of the native hph coding region.   Plasmid tgCMV / h was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. ygro-CD was constructed: The long chain SpeI-BstE2 fragment from tgCMV / CD was cloned into tgCMV / hyg a SpeI-ScaI fragment containing the hph coding region from ro / LTR, and Adult DNA fragment (is an oligonucleotide 5'-ACTCTCGAATAACGCTTTACAAACAATTATTAACGCCCG-3 'and Annealing 5'-GTAACCGGGCGTTAATAATTGTTTGTAAAGCGTTATTCGAGAGT-3 ' Prepared by).   Plasmid tgCMV / C was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. D-neo was constructed: a long ClaI-Asp718 fragment from tgCMV / CD was synthesized into a DNA fragment. Segment (is an oligonucleotide 5'-CGATTACAAACGTATTGAACAAGATGGATTGCACGCAGGTTCTCC-3 'and Anneal 5'-GGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATACGTTTGTAAT-3 ' Of the neo gene coding region from tgCMV / neo. It was ligated to the EagI-Asp718 fragment containing the rest.   Plasmid tgCMV / n was prepared using standard procedures (Ausubel et al., Supra, 1987) as follows. eo-CD was constructed: The long SphI-SalI fragment from tgCMV / neo was Is leotide 5'-CGAACTGTTCGCCAGGCTC-3 'and 5'-CCCGGTAACCGGGCGTTAATAATTGTTTGTAAAGCGTTATTCGAGAAGAACTCGTCAAGAAGGC-3 ' ClaI-NcoI fragment amplified from tgCMV / neo by PCR used (PCR before ligation (The product was cut with SphI and BstE2), and the CD gene codein from tgCMV / CD Ligated to the BstE2-SalI fragment containing the rest of the coding region.   B.Dominant positive selection of cells containing the CD fusion gene   To demonstrate that the CD fusion gene encodes both neo and hph activities, NIH / The frequency of various plasmids conferring drug resistance in 3T3 cells was examined.   First, NIH / 3T3 cells, 10% calf serum (Hyclone), 2 mM L-glutamine, 50 U / ml Dulbecco's modified E supplemented with penicillin and 50 μg / ml streptomycin Glue medium (DMEM; obtained from Gibco Laboratories) at 37 ° C, 10% CO₂Supplemented with It was grown in a humid atmosphere. Exponentially growing cells were harvested for transfection. Recovered by pushin treatment, washed to remove serum, and concentrated to 10⁷In DMEM at cells / ml Resuspended. Plasmid DNA (5 μg) was added to 800 μl of cell suspension (8 x 10⁶Cells) Then, this mixture was mixed with Biorad Gene Pulser and Capacitance Extender (200-3 00V, 960μF, 0.4cm electrode gap, ambient temperature) for electroporation I took it.   After electroporation, return the cells to a 10 cm dish and in non-selective medium. Grew. After 24 hours, cells were trypsinized and 6x10^FiveIn a cell / 10 cm dish Inoculated and contacted overnight I let you. Transfer non-selective medium to selective medium (containing 500 U / ml Hm or 800 μg / ml G-418). Replaced and continued selection for 10-14 days. The plate is then fixed with methanol , Stained with methylene blue, and colonies were counted. Colonies reported in Table 1 The number of colonies is the average number of colonies per 10 cm dish.   Non-transfected cells were hygromycin resistant (Hm^r) But G-418 resistance (G -418^rIt wasn't. From this result, hygro-CD fusion gene and CD-hygro fusion gene The gene is Hm^r, The activity of the CD-hygro fusion gene is Is lower than the activity of. CD-neo fusion gene is G-418^rGive, but neo- CD gene is not given.   C.Cytosine deaminase assay on transfected cell pools   To investigate whether the fusion gene retains cytosine deaminase (CD) activity For this purpose, Hm shown in Table 1^rAnd G-418^rPool and establish NIH / 3T3 colonies The cell line. Prepare the extract, and then [^ThreeH] -Instead of cytosine [¹⁴C] -Sitoshi As described previously (Mullen et al., Proc. Natl. Acad. S). ci. USA 89:33, 1992), to determine the conversion of cytosine to uracil. More CD activity was assayed. 1 x 10 in a 10 cm dish⁶Inoculate cells and cells 2 Incubated for days. The cells were then placed in Tris buffer (100 mM Tris, pH 7.8, 1 mM EDTA). , 1 mM dithiothreitol) and 1 ml Tris buffer. I scraped it off. The cells were then centrifuged in an Eppendorf microfuge for 10 seconds at 24,000 rpm. Resuspend in 100 μl Tris buffer aliquots, and snap freeze and thaw for 5 cycles Was done. Centrifuge at 6,000 rpm for 5 minutes in the Eppendorf microfuge, and then collect the supernatant. Transferred to a Reina tube.   Check protein concentration in extracts using the Biorad Protein Assay Kit Was. Then 25 μl of cell extract (ie protein equivalent in a volume of 25 μl) was added to 1 μl of[¹⁴C] -cytosine (0.6 mCi / ml, 53.4 mCi / mmol; Sigma Chemical Co.), The reaction was then allowed to proceed for 1-4 hours at 37 ° C. Half of the reaction is then applied to the thin layer chroma Chromatogram and added to the chromatogram in a mixture of 86% 1-butanol and 14% water. Tograph was done. After unfolding, the thin-layer chromatogram is analyzed by Kodak X-OMAT AR X-ray Exposed to Rum for 8-14 hours. The results are shown in Figure 2.   From the results, CD-neo fusion gene, CD-hygro fusion gene, and hygro-CD fusion gene The offspring encoded CD activity, but the CD-hygro fusion gene and hygro-CD fusion gene activity were The sex was shown to be lower than the activity of the CD-neo fusion gene.                              Example 2Retrovirus vector containing neo or CD-neo selection fusion gene                       Construction and characterization   A.Construction of retrovirus vector   The retrovirus plasmids tgLS (+) neo and tgLS (+) CD-neo contain the following factors: 5 'LTR and MoMSV nucleotide 984 up to the PstI site (Van Beveren et al., Cell  27:97, 1981); from the PstI site at nucleotide 563 to nucleotide 1040 of MoMLV. Sequence (Shinnick et al., Nature 293: 543, 1981); neo or CD-neo coding region Fragments derived from tgCMV / neo or tgCMV / CD-neo containing regions, respectively; MoM Sequence of nucleotides 7764 to 3'LTR of LV (Shinnick et al., 293: 543, 1981). N from pML2d (Lusky and Botchan, supra, 1981) containing the bacterial origin of replication ruI-AlwNI fragment; pGEM1 (Promega Corp containing the β-lactamase gene .) Derived from AlwNI-AatII fragment.   Plasmid tgLS (+) was added using standard techniques (Ausubel et al., Supra, 1987) as follows. ) neo was constructed: First, the EcoRI-ClaI fragment from tgLS (+) HyTK was added to tgCM. EcoRI-Asp718 fragment from V / hygro, and synthetic DNA fragment (oligo Is a nucleotide Anneal 5'-GTACAAGCTTGGATCCCTCGAGAT-3 'and 5'-CGATCTCGAGGGATCCAAGCTT-3' Prepared by ringing) The plasmid tgLS (+) hygro was constructed by Then it extends to the hygro gene NheI-HindIII fragment is an oligonucleotide 5'-CCCGCTAGCCGCCGCCACCATGGGATCGGCCATTGAACAAGATGGATTGCAC-3 'and 5'-CCCAAGCTTCCCGCTCAGAAGAACTCGTC-3 '(PCR product with NheI and HindIII before ligation PSV2neo (Southern and Berg, J. Mol. Appl. Gen. 1: 327, 1982) to replace the amplified NheI-HindIII fragment. Therefore, the plasmid tgLS (+) neo was constructed.   Plasmid tgLS (+) was added using standard techniques (Ausubel et al., Supra, 1987) as follows. ) CD-neo was constructed: HCV IE94 promoter from HyTK-CMV-IL2 and human IL-2  The NheI-SalI fragment spanning the cDNA was labeled with the NheI-SalI fragment from tgCMV / CD-neo. I replaced it with   Figure 3 shows the retrovirus vectors tgLS (+) neo and tgLS (+) CD-neo. The rovirus structure is shown. In the figure, "LTR" is the long-term anti-reverse of the retrovirus vector Represents a rearrangement segment, where "neo" is the bacterial neomycin phosphotransferase Ze gene, and "CD-neo" is CD / neomycin phosphotransferase ZE fusion gene is shown. neo gene and CD-neo gene operate in LTR transcription control region It is connected as possible. The arrow indicates the direction of transcription from the transcription control region. "A⁺"Indicates a polyadenylation sequence.   B.Passage of stable cell lines infected with retroviral vectors   To obtain stable NIH / 3T3 cell lines infected with tgLS (+) neo and tgLS (+) CD-neo In addition, the retroviral plasmid DNA of Ψ2 ecotropic packaging cells Transfected into. Next, the transfected Ψ2 cells were treated with 10 mM butyric acid. 10 cm tissue culture containing complete growth medium supplemented with sodium (Sigma Chemical Co.) Transferred to a petri dish and contacted overnight. After 15 hours, remove the medium, and Replaced with fresh medium. After another 24 hours, the temporarily produced ecotrophic window Collect the medium containing the virus particles, centrifuge at 2000 rpm for 10 minutes, and NIH / 3T3 Used to infect cells.   Exponentially dividing NIH / 3T3 cells were harvested by trypsinization and 2.5 x 10^FourFine Cells inoculated into two 6-well tissue culture trays at a density of cells / 35 mm well. Next day, medium Was supplemented with 4 μg / ml polybrene (hexadimethrine bromide) (Sigma Chemical Co.) Replaced with serial dilutions of cell-free supernatant (1 ml / well) containing virus in the culture medium . The infection was allowed to proceed overnight. The supernatant was then replaced with complete growth medium. Further 8-2 After 4 hours of growth, infected NIH / 3T3 cells were tested for drug resistance to G-418 (Gibco), Final concentration 800 μg / ml (Hm^rCells). 1 tray after growing for 12-14 days in total Culture of G-418^rResistant cells were fixed with 100% methanol and stained with methylene blue. Colored. Count the colonies and use the colony count in each well to give a temporary infection. The titer of the retrovirus present in the supernatant was established (Table 2).   Other tray G-418^rFrom cells, G-418^rPool cell colonies and analyze Bulk culture was established for. An extract was prepared from this bulk culture, and [^ThreeH] -Instead of cytosine [¹⁴C] -It has already been described in general except that cytosine was used. (Mullen et al., 1992), by measuring the conversion of cytosine to uracil. CD activity was assayed. 1 x 10 in a 10 cm dish⁶Inoculate cells and Incubated for 2 days. The cells were then treated with Tris buffer (100 mM Tris, pH 7.8, 1 mM ED TA, 1 mM dithiothreitol), and petri dish with 1 ml Tris buffer. I wiped it off.   The cells were then centrifuged at 14,000 rpm for 10 seconds in an Eppendorf microfuge and 100 μl Resuspend in Tris buffer and perform 5 cycles of deep freezing and thawing. Eppe Centrifuge at 6,000 rpm for 5 minutes in the ndorf microfuge, and then clean the supernatant into a clean tube. Moved to. Extract using Biorad Protein Assay Kit The protein concentration inside was examined. Then 25 μl of the cell extract (i.e. in a volume of 25 μl 1 ml of protein equivalent)¹⁴C] -cytosine (0.6 mCi / ml, 53.4 mCi / mmol; Sigma Ch emical Co.) and the reaction was allowed to proceed for 1-4 hours at 37 ° C. 2 minutes of reaction 1 was then added to the thin layer chromatogram, and 86% 1-butanol and 14% water were added. Was chromatographed in a mixture of After development, the thin-layer chromatogram was Exposed to OMAT AR X-ray film for 8-14 hours. From the results shown in FIG. 4, tgLS (+) CD-n Cells infected with the eoretroviral vector show high levels of cytosine deaminase. It is shown to express activity.   C.Negative selection of cells containing the CD-neo selection fusion gene   To examine the usefulness of neo and CD-neo selective fusion genes for negative selection In order to pool the colonies obtained from each transfection and A cell line was established for analysis. NIH / 3T3 cells or tgLS (＋) neo retrovirus Or NIH / 3T3 cells infected with tgLS (+) CD-neo retrovirus 5-FC using the assay^sAssayed.   First, 1 × 10^FourInoculate cells into a 10 cm tissue culture dish in complete growth medium and And contacted for 4 hours. The medium is then mixed with various concentrations of G-418 and / or 5-FC (Sigma ) And then incubated the cells for a further 10-14 days. Medium is 2-4 I changed it every day. The cells are then in situ with 100% methanol. Fixed and stained with methylene blue.   A photograph of a representative stained plate is shown in FIG. Plate a is in medium without drug NIH / 3T3 cells grown in. Plate b contains 800 μg / ml G-418 Have NIH / 3T3 cells grown in medium. Plate c contains 100 μg / ml 5-FC. NIH / 3T3 cells grown in culture medium. Plate d is tgLS (+) neo Have NIH / 3T3 cells infected and grown in medium containing 800 μg / ml G-418 . Plate e was infected with tgLS (+) neo 800 μg / ml G-418 and 100 μg / ml 5- Have NIH / 3T3 cells grown in medium containing FC. Plate f is tgLS (+ ) NIH / 3T3 cells infected with CD-neo and grown in medium containing 800 μg / ml G-418. With vesicles. Plate g was infected with tgLS (+) CD-neo to 800 μ / ml G-418 and Have NIH / 3T3 cells grown in medium containing 100 μg / ml 5-FC.   From these results, 1) non-infectious NIH / 3T3 cells were sensitive to G-418 and 5-FC 2) tgLS (+) neo-infected NIH / 3T3 cells were resistant to both G-418 and 5-FC. 3) tgLS (+) CD-neo-infected NIH / 3T3 cells are resistant to G-418 but 5-FC It is shown to be sensitive.

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Claims (1)

[Claims]   1. Is in reading frame with a negative selection gene and the negative Selectable fusion gene containing a dominant positive selection gene fused to Therefore, positive and negative selection phenotypes predominant in host cells when expressed A selective fusion gene encoding a single bifunctional fusion protein conferring an alternative phenotype ; Where the negative selection gene is cytosine deaminase (CD).   2. The dominant positive selection gene consists of hph gene and neo gene The selected fusion gene according to claim 1, which is selected from the group.   3. The selection fusion according to claim 2, wherein the dominant positive selection gene is neo. Syngene.   4. It encodes the sequence of amino acids from position 2 to position 690 set forth in SEQ ID NO: 2. The selective fusion gene according to claim 3, wherein   5. The nucleotide sequence from the 4th position to the 2073rd position of SEQ ID NO: 1 is coded. The selective fusion gene according to claim 3, wherein   6. A recombinant expression vector containing the selective fusion gene according to claim 2.   7. A recombinant expression vector containing the selective fusion gene according to claim 3.   8. A recombinant expression vector containing the selective fusion gene according to claim 4.   9. The recombinant expression vector according to claim 6, wherein the vector is a retrovirus. Tar.   10. The recombinant expression vector according to claim 7, wherein the vector is a retrovirus. Kuta.   11. The recombinant expression vector according to claim 8, wherein the vector is a retrovirus. Kuta.   12. A cell into which the recombinant expression vector according to claim 6 has been introduced.   13. A cell into which the recombinant expression vector according to claim 9 has been introduced.   14． Give cells a dominant positive and negative selection phenotype A method comprising introducing the recombinant expression vector according to claim 6 into the cell. Including, methods.   15. Give cells a dominant positive and negative selection phenotype A method comprising introducing the recombinant expression vector according to claim 9 into the cell. Including, methods.