JP2000500013A - Stable packaging cell line producing pseudotype retrovirus - Google Patents

Abstract

(57) SUMMARY The present invention relates to a stable pseudotyped retrovirus packaging cell line containing a packaging cell line that produces a helper-free recombinant pseudotyped retrovirus. The packaging cell line is under the control of (a) the retroviral gagpol gene and (b) an inducible operator system, the gene product of which controls the expression of non-retroviral genes that pseudotype the retroviral core virion. One or more non-retroviral expression constructs, such as the human cytomegalovirus (CMV) immediate early promoter or an expression construct having a derivative of this promoter (such as pMD). Furthermore, the present invention relates to a method for producing a stable pseudotyped retrovirus packaging cell line that produces a helper-free recombinant pseudotyped retrovirus. Furthermore, the present invention provides for the production of the special packaging cell lines described herein (ie, H29 gagpol, H29 new gagpol) and the stable pseudotyped retrovirus packaging cell lines described herein. (H29 cells and pMD, pMDtet, pMDtet.G, PMD.gagpol, pMD.new gagpol constructs, etc.). The present invention relates to a retroviral vector for the generation of a cDNA library for expression in mammalian cells containing two retroviral long terminal repeats, a cloning site for insertion of the cDNA and a cytomegalovirus promoter. The present invention also relates to a cDNA library for expression in mammalian cells, containing the retroviral vector of the present invention. The present invention also relates to a method for expression cloning in mammalian cells. The present invention also relates to a method for cloning cDNA expression in mammalian cells. The invention also relates to a method of identifying a deficiency in a gene responsible for a mutated phenotype using cDNA expression cloning by complementation in mammalian cells.

Description

DETAILED DESCRIPTION OF THE INVENTION     Stable packaging cell line producing pseudotype retrovirus Related application   This patent application is filed with Daniel S. Auri, Michael Saedrain and Lichi Chard Sea. Gene Transfer, filed November 8, 1995 by Mulligan Packaging cell lines producing pseudotyped retroviruses for use No. 08 / 555,155 and Daniel. S. Auri and Gene E. Released by Shaffer on May 21, 1996 The desired "Vesicular stomatitis virus-G for expression cloning by complementation ( VSV-G) Preparation of Retrovirus cDNA Expression Library Having Host Range Patent Application Serial No. 08 / 651,050 entitled "The Law" It is. Each teaching is incorporated herein by reference in its entirety. .Statement of fund   The work described herein was funded by the National Institutes of Health, K11 HL02910. More helped. The United States Government has certain rights in the invention.Background of the Invention   Recombinant retroviruses provide in vivo and in vitro gene expression and In addition, it is useful for producing a target protein in eukaryotic host cells. Generally, recombinant leto The rovirus is a suitable proviral DNA vector that encapsulates the desired recombinant RNA. That produce the viral proteins required for the production of recombinant and infectious recombinant virus particles It is produced by introducing into mammalian cells. For most gene transfer applications Therefore, a high-purity strain of replication-free helper virus-free recombinant virus Since the production of tocks is desired, the viral residues necessary for the production of recombinant retroviruses Produces a gene product, but does not itself produce a detectable helper virus, or Has considerable interest in developing cell lines that do not transmit viral genes (Co ffin, J., RNA Tumor Viruses, Weiss, R.A. Et al. Cold Spring Harbor La boratory, 2: 36-73 (1985); Mann, R. et al., Cell, 33: 153-159 (1983);  Watanabe, S. et al., Mol. Cell Biol., 3: 2241-2249 (1983); D. PN AS, USA, 81: 6349-6353 (1984); Miller, A .; D. Et al., Mol. Cell Biol., 6: 2895- 2902 (1986); Bosselman, R .; A. et al., Mol. Cell Biol., 7: 1797-1806 (1986). Or One approach to achieving this is to use a mutated proviral genome To develop a retrovirus packaging cell line. However , Helper virus production and / or packaging functions (ie, (Lus gene) transmission may still occur.   Therefore, an improved retrovirus that limits the ability to produce helper virus A packaging cell line is required.Summary of the Invention   The present invention produces a helper-free pseudotype retrovirus, Of animal origin, preferably of non-murine origin, such as a stable packaging human cell line. One stable packaging cell line. These are referred to herein, respectively. Stable pseudotyped retroviral packaging cell lines and stable pseudotypes It is referred to as an id-type retrovirus packaging human cell line. Package Cell line is a) a retrovirus, referred to as the retrovirus gagpol gene. gag gene and retroviral pol gene, and b) inducible operet Gene products that control the retroviral core virions. Human cytomega governs the expression of pseudotyping nonretroviral genes Lovirus (CMV) immediate early promoter or derivative of this promoter (Eg, pMD) containing one or more non-retroviral expression constructs. gagpo The l gene product can be obtained by pseudo-recombining the desired recombinant RNA with a non-retroviral gene product. Packaged in core virus particles that are id-typed and helper-free A stable pseudotype retrovirus patch capable of producing recombinant retrovirus. A caging cell line results.   In one embodiment, the invention provides a helper-free set having a pan-affinity host range. Stable pseudo-type leto capable of producing recombinant pseudo-type retrovirus Pertaining to rovirus packaging cell lines. These cell lines are helper-free Produce a recombinant pseudotyped retrovirus. Packaging cells The retroviral gagpol gene and vesicular stomatitis virus G (VSV- G) one or more non-retroviral expression constructs that govern the expression of the glycoprotein gene contains. Under the control of an inducible operator system (tet operator, etc.) The VSV-G glycoprotein is a retroviral gene produced by the gagpol protein. Provided is an envelope protein that pseudotypes the virus particles. The result The result is a helper-free recombinant pseudotyped retrospective with a pan-affinity host range. Stable pseudotyped retroviral packaging cells producing virus Strain (H29 gagpol, etc.). In another embodiment, the altered (mutation Etc.) Using a retrovirus gagpol gene, stable pseudo-type reto Rovirus packaging cell line (H29 new gagpol cell line, etc.) To manufacture.   The present invention further provides a helper-free recombinant pseudotyped retrovirus. Of a stable pseudotype retrovirus packaging cell line that produces Escherichia coli About the method. In the method of the present invention, the mammalian cell is subjected to a) retrovirus g agpol gene, and b) under the control of an inducible operator system, Provides pseudotyped envelope for rovirus core virions One or more non-retroviral expression constructs that govern expression of non-retroviral genes To transfect simultaneously. The gagpol protein is a non-retroviral gene The desired recombinant RNA is contained within the core virus particle pseudotyped by the product. Packaging and helper-free recombinant pseudotyped retrovirus Produces a stable pseudotyped retroviral packaging cell line that produces .   In one aspect, the invention relates to a helper-free, pan-affinity host range. Stable pseudotyped leto producing recombinant pseudotyped retrovirus The present invention relates to a method for producing a rovirus packaging cell line. In this embodiment, The animal cells are transformed with the retroviral gagpol gene (eg, wild-type or altered). ) And one or more non-retroviral expression constructs that govern expression of the VSV-G gene. Simultaneous transfection with a building. V under control of an inducible operator system The SV-G gene is the core of the retrovirus produced by the gagpol protein. It provides a pseudo-type envelope protein for virus particles. to this More Helper-Free Recombinant Pseudotype Leto with Pan-Affinity Host Range Stable pseudo-type retroviral packaging cells that produce virus This gives rise to a cell line.   In another aspect, the invention relates to a helper-free set having a pan-affinity host range. Stable pseudotyped retrovirus producing recombinant pseudotyped retrovirus The present invention relates to a method for producing a virus packaging cell line. In the method, the mammal The host cell is transfected with the tet transactivator fusion protein (tTA) gene. The first non-retroviral construct to appear (Gossen, M. and Bujard, M., Proc. Natl. Acad. Sci., 89: 5547-5551 (1992)) and the tet operator (tet bond VSV- under control of the smallest human CMV immediate-early promoter that incorporates the sequence) Simultaneous tracing with a second non-retroviral construct expressing the gene for the G glycoprotein Transfect. Transfected cells were transformed with tetracycline-inducible VS Screen for VG expression. VSV-G protein is tetracycline It is not detected in the presence of thiophene but is detected in the absence of tetracycline. Take The cell is treated with a third non-retrovirus expressing the retroviral gagpol gene. Transfect with the construct and screen for retrovirus production. Transfected cells that produce retrovirus have a pan-affinity host range. Stable, producing a helper-free recombinant pseudotyped retrovirus Pseudo-type retrovirus packaging cells.   In addition, the present invention relates to special packaging cell lines (H2 9 gagpol or 293GPG cell line, H29 new gagpol cell line Cell lines) as well as the stable pseudotyped retroviral proteins described herein. Packaging cell line (H29 cells and pMD, pMDtet, pMDtet.G, PMD. gagpol, pMD. new gagpol construct) Specific cells and constructs (such as packaging elements).   Another aspect of the invention is to provide a cDNA library for expression in mammalian cells. This is a retrovirus vector to be prepared. The retroviral vector comprises Two retroviral long terminal repeats (LTRs) (5 'retroviral LTR and 3' Cloning site for insertion of cDNA and It contains the Itomegalovirus (human etc.) promoter. In one embodiment, Two LTRs are the Moloney murine leukemia virus (MMLV) . In specific embodiments, the 3 'MMLV LTR is unmodified and the 5' is modified Or the U3 region of the 5 'MMLV LTR is cytomegalovirus (human ) Or cytomegalovirus enhancer-promoter Is a chimeric MMLV LTR substituted with   The present invention also relates to a cDNA library for expression in mammalian cells. You. The library contains two retroviral LTRs, cDNA and cytome Contains a retroviral vector containing a gallovirus promoter. The c The DNA is located at a unique cloning site in the retroviral vector, Operably linked to the cytomegalovirus promoter located between the LTRs of Is preferred.   The present invention also relates to a method for expression cloning in mammalian cells. The method is A cDNA expression library containing the retroviral vector of the present invention. Step of introducing into an animal cell, and conditions suitable for expression of the cDNA expression library And maintaining a mammalian cell containing said expression library under said conditions. Expressing the cDNA in the library in said mammalian cells. One aspect In the above, the cDNA expression library is transformed into a stable mammalian (human, murine, etc.) Infect with a pseudotyped retrovirus produced in a packaging cell line Thereby, it is introduced into mammalian cells. Stable mammalian packaging cell line Select, pan-affinity, ecotropic or amphotropic host range, preferably Produces a pseudotype retrovirus with a pan-affinity host range. special In another aspect, the invention relates to a method for expression cloning in a mammalian cell. You. The method comprises two retroviral LTRs, a cDNA and a cytomegaloyer. CDNA expression vector containing retroviral vector containing The library is used for packaging pseudotyped retrovirus-producing cells. Introducing into a strain; a pseudo-type retro containing the cDNA expression library A package containing the expression library under conditions suitable for the production of virus particles. Maintaining the cell line; pseudotypes under conditions suitable for mammalian cell infection. Infecting a mammalian cell with a retroviral particle of the mammal; and the mammal Maintaining the resulting mammalian cell under conditions suitable for expression of the cDNA in the cell. including. In a specific embodiment, the packaging cell line is a stable human fetal kidney cell Strains, and specifically cell lines derived from human 293.   In addition, the present invention provides a method for producing a cDNA library by transcription of cDNA. VSV-G pseudotyped retroviral particles containing RNA Expression cloning method of cDNA in mammalian cells produced by Zing cell line About. The cDNA library in turn contains two retroviral LTRs, contains a vector containing cDNA and cytomegalovirus promoter The cDNA is operably linked to a promoter located between the LTRs; . The mammalian cells are then placed in a VSV-G pseudotyped retroviral particle. Transcription of contained RNA and cDNA in cDNA library in mammalian cells Of a protein encoded by (translation of RNA in retroviral particles) VSV-G pseudotyped retroviral particles produced under conditions suitable for Infect. RNA contained in VSV-G pseudotype retrovirus particles Or a mammal containing a protein encoded by a cDNA in a cDNA library. The target cells are detected using a known method. For example, RNA is in situ It can be detected using hybridization. Alternatively, if the cDNA is Encodes a protein expressed on the cell surface, and expresses the expressed protein with the target cDNA. Uses immunodetection that can be detected using antibodies that bind to the expressed protein (See, eg, US Pat. No. 5,506,126). Ma In addition, the expressed protein can be detected using the epitope tag. Also the machine The function of the protein expressed by the cDNA of interest (adhesion table in cells) Protein that gives the present type) can be detected.   The present invention also provides cDNA expression clonin by complementation in mammalian cells. The present invention relates to a method for identifying a gene defect that causes a mutant phenotype by using the method of the present invention. This one In the method, RNA produced by transcription of cDNA in a cDNA library is Containing VSV-G pseudotyped retroviral particles in a packaging cell line Produced. The cDNA library contains two retroviral LTRs, cDN A and the cytomegalovirus promoter; the cDNA contains the LTR It is operably linked to a promoter located in between. Mutated phenotype Milk cells were isolated from RNV contained within a VSV-G pseudotyped retroviral particle. A transcribed and encoded by the cDNA in the cDNA library (retro Under conditions suitable for the production of proteins (by translation of RNA in virions), VSV -Infect with G pseudotyped retroviral particles. When the cDNA is expressed Mammalian cells with a mutant phenotype exhibiting a wild-type phenotype are identified. Then Identified a cDNA that confers a wild-type phenotype in mammalian cells, and Determine the gene deficiency that causes the heterophenotype.   Stable pseudotyped retroviral packaging described herein Cell line development suppresses the generation of helper virus. As a result, a stable Id-type retroviral packaging cell lines are used for cell lineage analysis and In vivo gene transfer research aimed at developing gene replacement therapy It is a useful reagent for   The use of the retroviral vectors of the present invention can be used in any mammalian cell type. Enables efficient cloning of retrovirus cDNA Avoid the need for specialized cells for training. Different from wild-type parent cell type Any mutant mammalian cells in which a phenotype is present (primary human cells from patients, sudden A primary or established cell line derived from a mutant animal strain, a primary cell line derived from a knockout mouse Or established cell lines, mutant cell lines produced in cell culture, etc.) Genetic differences (mutation or deletion) between mutant and wild-type cells Can be rapidly identified by expression cloning by complementation using the present invention.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic of the pMD construct.   FIG. 2 is a schematic of the pMDtet construct.   FIG. FIG. 2 is a schematic diagram of a G construct.   FIG. 4 shows pMDtet. FIG. 2 is a schematic diagram of a G construct.   FIG. FIG. 2 is a schematic diagram of the gagpol construct.   FIG. FIG. 2 is a schematic diagram of the new gagpol construct.   FIG. 7 shows plasmid pBC. It is a schematic diagram of tTA.   FIG. 8 shows plasmid MFG. It is a schematic diagram of SnlsLacZ.   FIG. 9A is a schematic of the ΔU3 retroviral construct ΔU3nlsLacZ. .   FIG. 9B is a schematic of the ΔU3 retroviral construct ΔU3Bam.Detailed description of the invention   The present invention produces pseudotyped retroviruses of mammalian origin, A stable retrovirus, preferably of non-murine origin, such as a stable packaging human cell line. It relates to a virus packaging cell line. These are referred to herein, respectively. Produces pseudotyped retroviruses for retroviral gene transfer Stable pseudotyped retroviral packaging cell lines and stable pseudotypes It is referred to as an id-type retrovirus packaging human cell line. The package of the present invention Caging cell lines are retroviruses that produce retroviral core virus particles. Psoi against the gagpol protein and retroviral core virions One or more non-retroviruses for expressing a protein that provides a Contains the Ils construct. Pseudo-type for retroviral core protein The protein that provides the envelope is under the control of an inducible operator system. That is, retroviruses by the packaging cell lines described herein. Production is a pseudotype enveloped against the retroviral core virus particles. Controlled by the inducible expression of the protein that provides the protein. The package of the present invention Cell lines are non-induced (eg, when using the tet operator, Viable, non-enveloped retrovirus (in the presence of lacyclin) Expresses the gagpol protein; the underivatized packaging cell line was induced Case (eg, when using the tet operator, the absence of tetracycline) Below), capable of producing (producing) recombinant pseudotyped retroviral particles . Thus, the pseudotype retroviral packaging cell line of the present invention Is stable. Once induced, the packaging cell line is a recombinant pseudotype Produces (produces) ip retroviral particles.   Further, the packaging cell line of the present invention suppresses the ability to produce a helper virus. You. For non-retroviral constructs and retroviral core virions, The use of nonretroviral proteins that produce pseudotyped envelopes is Contributes to the suppressed production of parvirus. Helper virus producing ability It can be further suppressed by using myocytes (such as human cells). Retro virus To generate a packaging cell line, a murine cell line (such as NIH3T3 cells) Is typically used. However, in the case of murine cell lines such as NIH3T3 cells, Presence of endogenous murine retrovirus in nom (Danos et al., Proc. Natl. Acad. Sci. , 85: 6460-6466 (1988)), the host cell genome, retroviral expression constructs and Facilitate the recombination event between the virus and the retroviral vector, thereby helper virus Contributes to the production of water.   Further, by changing the retrovirus gagpol gene (mutation, etc.), The ability to produce Ruper virus can be limited. Mutated gagpol Example sequence (i.e., new 5 'gagpol; new 3' gagpol) Is described in Example 1.   As described in Example 2, the packaging cell line of the present invention was Novel non-retroviral human CMV immediate early promoter for expressing genes Expression Construct (pMD) and Pseudo Inhibiting Helper Virus Production Ability Can be derived from human 293 cells incorporating a type of envelope . Also, silent mutations in the gagpol coding sequence can be The homology with the vector sequence, and further enhance the ability to produce helper virus. Suppress. As further described herein, a packaging cell line of the invention Is a vesicular mouth that efficiently pseudotypes retroviral core virions It expresses the glycoprotein of endositis virus G (VSV-G). The glycoprotein of VSV-G is Has a broad host range. Therefore, the pseudo-type reto with VSV-G Viruses exhibit a broad host range (pan-affinity), ecotropic and amphotropic Cells that are resistant to tropic retrovirus infection can be efficiently transmitted. (Yee, J.-K., et al., Proc. Natl. Acad. Sci., 91: 9564-6568 (1994)). VS The high level expression of VG is cytotoxic, and therefore a novel packaging cell Expression of VSV-G in the strain is determined by inducible expression such as an inducible tet operator system. It is controlled by the pererator system and inherited by the concentration of tetracycline in the culture medium. Allows for tight regulation of offspring expression (ie, production of retroviral particles).   Lastly, as shown herein, pseudotyped reticulum with VSV-G Lovirus particles can be concentrated 100 times or more by ultrafiltration (Burn s, J .; C. et al., Proc. Natl. Acad. Sci., 90: 8033-8037 (1993)). Stable VSV 1 ml of the retrovirus packaging cell line pseudotyped with -G 10 per⁷-10¹¹A range of retroviral particles produces a retroviral stock solution Large-scale virus preparation (eg, 10-50 liters of supernatant) I do.   H29 cells expressing inducible VSV-G protein had more than 20 passages. Cell cultures. H29 cells at passage 20 remain viable, VSV-G detectable in an inducible manner at levels comparable to cells at early passage Expressing proteins (eg, by cell fusion studies, Western blotting, etc.) to continue.   The expression construct used in the present invention produces a retroviral core virus particle. Gagpol gene and retrovirus gene used for Expression of a protein that provides a pseudo-type envelope for virus particles Is a non-retroviral vector that governs As described in Example 1, the book Expression constructs suitable for use in the invention include human cytomegalovirus (CMV) immediate- Early promoter construct. Other constructs that can be used to practice the invention Examples of using SV40, RSV and rat β-actin promoters Construct.   Known in the art using one or more non-retroviral expression constructs Of gagpol gene and pseudo-type envelope using the technology of Can be expressed. For example, the protein may be It can be expressed using a Lus expression construct. Also, one construct is gag Under the control of an operator that expresses the pol gene and is inducible by other constructs, Expressing a gene (VSV-G, tTA) that provides an envelope of One non-retroviral expression construct can be used. Alternatively, Example 1 Three non-retroviral constructs: the first non-retroviral as described in The construct is an inducer that controls the expression of a gene that expresses a pseudotype envelope. Encoding a transducible tet transactivator protein (tTA); Non-retroviral constructs contain genes that provide a pseudotyped envelope. Express and the third non-retroviral construct expresses the gagpol gene To be used. Furthermore, the gag and pol sequences are generated separately. Which can be expressed by a fourth non-retroviral construct (eg, A third retroviral construct expresses the gag gene and a fourth retroviral origin Current construct expresses the pol gene).   As referred to herein, a "pseudo-type envelope" is A retroviral core virus that encapsulates the viral core virus particles in a capsid Envelope proteins other than those naturally occurring in microparticles (phenotypically mixed). If the virus is present). Suitable proteins that provide a pseudo-type envelope Is the vesicular stomatitis virus G (VSV-G) glycoprotein as described in Example 1. Quality. In the present invention, any suitable serotype of VSV-G (India Na, New Jersey, chandipura, pili etc.) and strains (V SV Indiana, San Juan, etc.) can also be used. core Proteins selected for pseudotyping virions are packaged Determine the host range of the cell line. VSV-G is a specific receptor on the surface of mammalian cells. Interacts with phospholipids (Schlegel, R. et al., Cell, 32: 639-646 (1983); Supertzi J., F. et al. Gen Virol., 68: 387-399 (1987)). Therefore, use VSV-G Pseudotyped envelope for retroviral core virions Packaging cell lines that have a broad host range (pan-affinity). Leto Provides pseudo-type envelope for core virions of rovirus Other suitable proteins that can be used for purification include murine C retrovirus HTLV-1 envelope protein, gibbon (Gibbo) n ap) Leukemia virus envelope protein and pseudo-type envelope Derivatives of suitable proteins that provide the rope (e. Targeting such as envelopes, synthetic and / or other hybrid envelopes Protein containing insertions, deletions or mutations for preparing modified envelope sequences VSV-G glycoprotein derivatives, etc.). In addition, the murine retrovirus envelope Derivatives of rope proteins can be used. For example, binding to the cell surface The VSV-G protein is replaced with a specific ligand to achieve the membrane fusion. Obtaining a derivative of VSV-G protein holding a practicable portion of VSV-G protein Can be Portions of the VSV-G protein that can effect binding to the cell surface include, for example, For example, determination by performing point mutation and deletion sequence analysis of the VSV-G sequence. Is done. The ability of each mutated VSV-G protein to bind to the cell surface is determined by the appropriate binding Determined by using Here, the derivative of the VSV-G protein was incorporated. Retroviral particles can be targeted to specific cell populations. You.   As discussed above, navigable operators are pseudo-type envelopes. It is used to control the expression of genes that provide loops. For example, a high level V The expression of SV-G is cytotoxic (Yee, J.-K. et al., Proc. Natl. Acad. Sci. , 91: 9564-6568 (1994)). Thus, inducible tetracyclines (ie, , Tet) Tet in the culture medium of the packaging cell line using the operator system The concentration of lacyclin allows tight regulation of VSV-G expression. Sand In the presence of tetracycline, tetracycline Clin binds to the tet transactivator fusion protein (tTA) and Control of tet operator sequence by preventing A from binding to tet operator sequence Under the gene expression (Gossen, M. and Bujard, M., Proc. Natl. Acad. Sci., 89: 5547-5551 (1992)). In the absence of tetracycline, tTA is It binds to the tet operator sequence to generate a gene under the control of the tet operator. Impede reality. Controls the expression of proteins that provide a pseudo-type envelope Examples of other inducible operator systems that can be used for 1) metal ions (meaning Induction in response to glucocorticoid hormone) A possible eukaryotic promoter and 2) the lac repressor / operator of E. coli. It is a lator / inducer system.   The nucleotide sequence encoded by the non-retroviral construct is used in the present invention. It can be obtained from various suitable sources used. For example, the operator system Expressed nucleotide sequence, pseudotyped envelope and gagpol Sequences can be purified from natural sources, produced by chemical synthesis, or used in recombinant DNA technology. Can be manufactured. For example, as described in Example 1, gagp The ol sequence is pCripenv^-It can be obtained using the construct.   Cells used to prepare the packaging cells may be mammalian cells, preferably Or non-murine cells. In a specific embodiment, a packaging cell line is produced The cells used to transform the cells are human cells (eg, 293 cells, Graham, F. et al. Gen. Virol., 36: 59-72 (1977); tsa201 cells, Heinzel, S .; Et al., J. Virol., 62: 3738 (1988)).   Using the packaging cell line of the invention, the desired gene can be expressed in eukaryotic cells. Genes can be expressed in vitro and in vivo for the purpose of expressing all or part of the gene. Can produce a recombinant pseudotyped retrovirus that allows transmission You. For example, using known techniques, the packaging cell lines described herein Using mRNA or protein in an amount useful for therapeutic administration or diagnostic situations To produce quality, a specific mRNA, protein or polypeptide (insulin , Human growth hormone, erythropoietin and other therapeutic proteins or polypeptides Pide, cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor Ter), ADA deficiency (ADA), Gaucher disease (glucocerebrosidase) Gene replacement, antisense therapy by expression of inhibitory mRNA sequences, etc.) Of the recombinant pseudotype used to introduce the gene to be introduced into eukaryotic cells A retrovirus can be produced (Yee, J.-K., et al., Proc. Natl. Acad. Sc. i., 91: 9564-6568 (1994); Dranoff, G .; Proc. Natl. Acad. Sci., 90: 353 9-3543 (1993); Miller, A .; D. Meth. in Enz., 217: 581-599 (1993)). sand In other words, a pseudo-type recombinant virus was recovered from a packaging cell line. Infecting recipient cells in culture or in vivo using known methods Can be used as a virus stock solution. In secreted proteins or hematopoietic cells For expressed proteins, high sensitivity such as ELISA or Western blotting Using a simple assay, the efficiency of gene transfer can be evaluated. Alternatively, The high titer virus stock solution produced by the packaging cell line contains transduced cells ( Provides superior gene transfer efficiency for hematopoietic cells, etc., compared to current co-culture technologies To reduce contamination.   In addition, using the packaging cell line of the present invention, a region localized in the body is subsequently used. A target DNA or gene to be administered into a mammalian cell such as a human cell To produce a pseudotype retrovirus containing the DNA of interest (Eg, for the delivery of proteins into localized areas (ares) of the body). Ex vivo infection of autologous transplanted leukocytes, US Pat. No. 5,399,346. See the specification).   We also generate stable pseudotyped retroviral packaging cells. The packaging element used to make it can be used in various ways . For example, using an H29 cell line showing inducible VSV-G expression, A retrovirus library for cleaning can be prepared. High titer The ability to produce viral stock solutions in Improve. Further, the packaging cell line of the present invention further comprises a pseudo-type It can also serve as a basis for the generation of a packaging cell line.   Efficient structural (such as pMD) and inducible (pMDtet) gene expression As an expression construct for the purpose, a packaging element can be used. For inducible expression of VSV-G, pMDtet. Use G-constructs for other applications Is possible. pMD gagpol and pMD new gag Use ol to Develop New Retroviral Packaging Cell Lines be able to. For efficient expression of heterologous genes, pMD, pMDtet, pMD MDtet. G and pMD. The gagpol construct can be used.   Thus, as described herein, VSV-G is generated in an inducible manner. The resulting stable cell lines have been generated. In addition, human genome β Novel CMV expression vector (pMD) using globin sequence and its derivatives ( pMD. G, pMDtet. G, pMD. gagpol, pMD. new ga gpol) was developed. Further, as described herein, CMV expression compared to a mutated proviral construct that suppresses the ability to A stable packaging cell line based on 293 using the construct has been developed and Mutated g in a stable cell line to suppress the capacity for The use of the agpol expression construct has been demonstrated.   The present invention also provides a clone for insertion of two retrovirus LTRs and cDNA. Mammalian site containing a cloning site and cytomegalovirus promoter. Retroviral vector for producing a cDNA expression library expressed in vesicles About. In one embodiment, the two LTRs are Moloney murine leukemia virus. Luz (MMLV). In a specific embodiment, the 3 'MMLV LTR is modified However, the 5 'is modified or the U3 region of the 5' MMLV LTR is not Itomegalovirus promoter or cytomegalovirus enhancer Chimeric MMLV LTR replaced with a promoter.   Retroviral LTRs can be derived from any suitable retroviral vector. A retrovirus that results in high titer or expression of a retroviral protein It is preferably a vector. The two LTRs are the same retroviral vector Or from different retroviral vectors. For example, the implementation As described in Example 3, both retroviral LTRs were Moloney murine leukemia. It may be derived from a virus (MMLV). Other suitable retroviruses LT As R, for example, murine sarcoma virus (MSV), murine papillary sarcoma virus (MPSV) and those derived from Friend virus.   The cloning site of a retroviral vector is a variety of cloning sites. You may. For example, as described in Example 3, the cloning site was BamH It may be an I cloning site. Use in retroviral vectors of the invention Other cloning sites suitable for, for example, gagpol or env Includes unique or rare restriction sites within the genome or U3 region .   Cytomegalovirus promoter can be obtained from any suitable source. It is possible to For example, as described in Example 3, complete cytomegalow The ils enhancer-promoter is a human cytomegalovirus (HCMV) Origin. Some or all of the previously described CMV promoters may be used in accordance with the invention. Can be used with To obtain the cytomegalovirus promoter Other suitable sources include commercial sources such as Clontech, Invitrogen and Stratagene. Sources.   The retroviral vector of the present invention is suitable for expression of a cDNA expression library. CDNA containing a retroviral vector described herein under conditions Expression clones in mammalian cells, wherein the expression library is introduced into the mammalian cells It can be used for In one embodiment, the present invention provides that the library comprises two libraries. Retroviral LTR, cytomegalovirus promoter and cDNA Containing the cDNA located between the retroviral LTRs CDNA expression for expression in mammalian cells operably linked to a promoter Regarding the library.   The cDNA used in the present invention is an object for expression in mammalian cells Any cDNA may be used. The cDNA may be a blood cell, a cell from a tissue sample or May be derived from any type of cell, such as a cultured cell. Generally, cDNA is When expression cloning by complementation is progressing, cDNA continues to be expressed. From the same type of cells.   The cDNA library used in the present invention can be prepared by a conventional method (Seed and Aruffo, Pr. oc. Natl. Acad. Sci, USA, 84: 3365-3369 (1987)). it can. For example, mRNA is prepared from any cell and cDNA is Techniques (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) and commercially available cloning Synthesizing using kits (Pharmacia, Invitrogen, Stratagene, etc.) Can be.   The retroviral vector of the present invention may be a cDNA expression library in a cell. Any technique that produces the expression of (electroporation, calcium phosphate precipitation , Cationic lipids, liposomes, etc.) can be introduced into mammalian cells. Wear. In one embodiment, the cDNA expression library is packaged in a cell line Containing the RNA transcribed from the cDNA expression library, Infecting mammalian cells resulting in expression of a cDNA expression library in mammalian cells Produce the retroviral particles used for In this embodiment, the package Cell lines (such as the 293GPG packaging cell line described herein) For expression cloning in mammalian cells as described in Example 4. Useful pseudotyped retroviral particles can be produced. In the present invention Other packaging cell lines suitable for use are derived from packaging cell lines Other human cell lines (eg, from embryonic cell lines) as well as Psi-2 cells (Mann, R . Cell, 33: 153-159 (1983); FLY (Cossett, FL et al., Virol., 193: 385-3. 95 (1993)), BOSC 23 cells (Pear, WS et al., Proc. Natl. Acad. Sci, USA 90: 8392-8396 (1993), PA317 cells (Miller, AD and C. Buttimore, Molec. . and Cell. Biol., 6: 2895-2902 (1986)), Kat cell line (Finer, MH et al., Bl. ood, 83: 43-50 (1994)), GP + E-86 cells and GP + EM12 cells (Markow itz, D., et al. Virol., 62: 1120-1124 (1988), and Psi Crip and Ps i Cre cells (US Pat. No. 5,449,614; Danos, O. and Mulliga) n, R. C. , PNAS, USA, 85: 6460-6464 (1988)) Murine cell lines. See also Young, Y. et al., Human Gene Ther., 6: 1. See also 203-1213 (1995). The packaging cell line used in the present invention is With a pan-affinity, amphotropic or ecotropic host range Ruth particles can be produced. Therefore, in this embodiment, the c DNA expression libraries are derived from retroviruses produced by packaging cell lines. It can be expressed in any cell within the host range of the Ruth particle. further, In this embodiment, the promoter of the retroviral vector of the present invention is a special promoter. Produces sufficient transcript levels of retroviral vectors in packaging cell lines Any promoter (SV40 promoter, RSV promoter, β- Actin promoter).   The retroviral vector of the present invention (ΔU3 retroviral vector, etc.) For production of titer pseudotyped retroviruses (such as VSV-G), pseudotyped Cell line producing a retrovirus of the same type (H2 herein). 293GPG cells and the like described herein, also referred to as 9 gagpol cells ) Can be used for transient transfection. Retrow of the present invention The ills vector is a pseudoretrotype containing a cDNA expression library. Transfection of retroviral packaging cell line producing irs particles Of cDNA expression library for cloning (retroviral vector At). Retrovirus particles of each pseudotype generally contain a large number of Includes mRNA molecules.   As described in Example 4, any cell line having a VSV-G host range ΔU3 retrovirus vector, which can be used for expression cloning by complementation Virus pseudotyped with high titers of VSV-G using Produced using GPG cells. For retroviral vector, ΔU3nlsLZ And 3x10⁶Retrovirus at titers up to infectious units (iu) / ml Was transfected. In addition, as described in Example 5, Using a retroviral cDNA cloning vector, ΔU3BAM 293GPG at the same expression level and viral titer as the ΔU3nlsLZ vector. Cells were transfected. 5 'non-expression in cDNA expression and virus titer Based on the evaluation of the effect of the translated sequence, the viral ΔU3Bam vector -Based Leto Optimized for High Expression and High Virus Titer Generation Only a modest reduction in expression or viral titer (compared to It shows that up to 165 base pairs of the 5 'upstream non-coding DNA sequence is adaptable Was. VSV-G reto produced by 293GPG cells described herein Lovirus pseudotypes have a broad host range and can be of any mammalian type. (Yee, J.-K. et al., PNAS, 91: 9564-9568).   Thus, the present invention can be used with a variety of mammalian packaging cell lines. And then the expression products (proteins, polypeptides) encoded by the cDNA Pseudo-type retrovirus that can be used to infect a variety of mammalian cells Provided is a retrovirus vector capable of producing an ils particle. Specific example smell Using a retroviral vector to transiently transfer 293GPG cells. To produce a high titer virus with a VSV-G host range. ΔU3 The BAM retroviral vector can be any vector from any cDNA library. Also allows for cloning into 29 of the retrovirus cDNA library Transfect 3GPG cells to detect any mammalian cell type 10 dyeable⁶i. u. Produces retroviruses with titers in excess of / ml. this Infection of the host cell with the retroviral vector is based on the cD in the retroviral construct. Stable integration of the proviral genome promotes long-term, high-level expression of NA Is generated.   The novel methodology described herein uses a retroviral vector (ΔU3Bam Torovirus vector, etc.) and packaging cell lines (293GPG cells, etc.) Utilization of retroviral cDNA expression clones in any mammalian cell type. Specialized cells (Cos7) to provide cloning and efficient expression cloning Cells, oocytes, etc.). Different phenotype from wild-type parent cell type Any mutated mammalian type present (primary human cells from the patient, from the mutated animal strain) Primary or established cell line, primary or established from knockout mice Cell lines, mutant cell lines produced in cell culture, etc.) Quickly identify deletions in one or more genes by gender-based expression cloning Can be   An extension of the methodology of the present invention is that cells from patients with genetic deficiencies and established phenotypes For the expression of candidate cDNA clones in packaging cells The retroviral vectors of the invention are used to produce retroviruses of the type It is to use. This allows for gene deficiency (alternative) in patients by complementation analysis. Screening to determine the basis for altered expression of genes associated with Xie Enable. For example, many patients have a single genetic defect in fatty acid metabolism. Has been genetically characterized biochemically. However, the mutation Has not been established by conventional methods. Various enzymes in fatty acid metabolism A panel of retroviral constructs encoding candidate cDNAs for, for example, Can be tested for complementation in primary fibroblasts from these patients You.   The retroviral vector of the present invention can be used for packaging cell lines (such as 293GPG). ) To promote high levels of expression in retroviral enhancer promoters. Is correctly replaced by the cytomegalovirus enhancer promoter (HCMV etc.) Retrovirus-derived vectors (Moloney murine leukemia virus Derived vectors).   Furthermore, any mammalian cells can be prepared using the retroviral vectors of the present invention. A retroviral cDNA expression library that also enables expression cloning in a template Can be produced. In addition, human cloning is performed by expression cloning by complementation. The identification of the mutated gene responsible for the gene deficiency was performed using the retroviral vector of the present invention. Can be achieved using Mutations induced in culture and having a known phenotype Primary or established cells derived from cell lines or animals with a mutated phenotype Expression cloning of a mutated gene from a cell line can be performed. For example, The expression cDNA library of the present invention is introduced into cells having a mutated phenotype, The identification of one or more genes that complement the loss is determined. Therefore, according to the present invention, An important advantage provided is that expression clones by complementation as described herein Using tanning to identify one or more genes responsible for the phenotype caused by the mutation Be able to identify and obtain evidence of the function of one or more causative genes That is. In addition, embodiments of the present invention provide gene products that complement an introduced mutation. Or knockout of a gene product that functions in one or more pathways affected by the mutation. In the Coutout mouse, the identification (ie, downstream F Identification using the retroviral vector of the present invention. Can be. In addition, retroviruses constructed with retroviral vectors for commercial sale A rovirus cDNA library is also provided.   The present invention will be described in more detail with reference to the following examples. It is not specified.Example Example 1 Construction of expression vector   pBC containing pXF3 backbone and human CMV immediate early promoter region 12. A 3.1 kb EcoRI-BamHI fragment from AB and B of exon 2 690 bp human β-globin genome sequence from the amHI site to the 3 'untranslated region PUCMdβs (R) S containing the sequence (Sadelain, M. et al., Proc. Natl. Acad. Sci. U SA, 92: 6728-6732 (1995)) and a 1.34 kb BamHI-XbaI fragment. Was used to construct pMD (see FIG. 1). However, the plasmid pUC Mdβs (R) S is 374b at the second intron between the first and third RsaI sites. It differs from the genomic sequence in that there is a deletion of p. pBC12. AB is I PBC12 replacing the L-2 sequence (bp 756 to 1439) with a polylinker / CMV / IL-2 (B. Cullen, Cell 46: 973 (1986)). Ec After the oRI and XbaI overhangs were blunt-ended by treatment with Klenow fragment, The 3.1 kb EcoRI-BamHI fragment and the 1.34 kb BamHI-Xba The I fragment was ligated.   derived from pSVGL (Rose and Bergman, Cell 34: 513 (1983)), human β-globin VS cloned into EcoRI site in pMD in exon 3 of genomic sequence Using a 1.6 kb EcoRI fragment containing the VG gene, pMD. G (Fig. 3 See).   pUHC 13- containing tet operator and minimal CMV promoter sequence 3 (Gossen and Bujard, Proc. Natl. Acad. Sci. 89: 5547-5551 (1992)). . 47 kb XhoI-BamHI fragment, 1.3 derived from pUCMdβs (R) S BamHI-XbaI fragment of 4 and 3.0 from pSL301 (Invitrogen). Using the 6 kb XbaI-XhoI fragment to generate pMDtet (see FIG. 2) did.   pMDtet. To construct G (see FIG. 4), the VSV G gene (pS VGL) and a 1.6 kb EcoRI fragment containing the human β-globin genomic sequence. PMD. It was cloned into the EcoRI site in tetG.   pMD. To construct gagpol (see FIG. 5), pCRIPenv- (Danos Mulligan, Proc. Natl. Acad. Sci. 85: 6460-6466 (1988)) and the following Immer vs: Was used to perform PCR. The PCR product was digested with EcoRI and XhoI and digested with EcoRI and HindIII, respectively, and digested with 0.94 kb of EcoRI-Xho. I and 0.94 kb HindIII-EcoRI fragments were generated. These cuts The piece was ligated with a 3.3 kb XhoI-HindIII fragment from pCRIPenv- and With pUC19 which had been linearized with EcoRI and phosphatase treated Ligation, pUC19. gagpol was prepared. pUC19. gagpol The next 5.2 kb EcoRI fragment was ligated to exon 3 of the human β-globin genomic sequence. Cloned into the EcoRI site of pMD within pMD. gagpol Obtained.   pMD. To construct new gagpol (see FIG. 6), the mutated g pBCIL2.coding the agpol sequence. gagpol (Chung and Mulligan, not yet Announcement results) and the following primer pairs: Was used to perform PCR. The mutated gagpol sequence is as follows: New 5 'gagpol: New 3 'gagpol:   The PCR product was digested with EcoRI and XhoI and EcoRI and HindIII. Each digested, 0.94 kb EcoRI-XhoI and 0.94 kb Hind A III-EcoRI fragment was generated. These fragments were obtained from pCRIPenv- A 3.3 kb XhoI-HindIII fragment and linearized with EcoRI It is ligated with pUC19 that has been subjected to a phatase treatment to give pUC19. new gag pol was produced. pUC19. 5.2 kb Ec from new gagpol The oRI fragment was transformed into the Ec of pMD within exon 3 of the human β-globin genomic sequence. cloning into the pRI. New gagpol was obtained.   Expression of wild-type gagpol (pMD.gagpol) and mutated gagpol ol (pMD. new gagpol), a novel CMV expression vector ( pMD) was constructed. pMD. For gagpol, both transient and stable Perform a reverse transcriptase assay that suggests retroviral particle production under current conditions. Was. pMD. For new gagpol, retrograde under conditions of transient expression A reverse transcriptase assay was performed indicating production of irs particles.Example 2 Construction of packaging cell line H29 cell line .   Human 293 cells (gift from B. Panning; Graham, F., et al., J. Gen. Virol., 36 : 59-72 (1977)) with 2 mM L-glutamine, penicillin and streptoma. DMEM (293 growth medium) containing 5% inactivated fetal bovine serum supplemented with isine Ground) and incubated at 37C, 5% CO2. The 293 cells 5 μm by the calcium phosphate precipitation method (Pear et al., PNAS, 90: 8392-8396 (1994)). g of PBC. tTA (see FIG. 7; T. Chung and R. Mulligan, unpublished results), 5 μg of pMDtet. G and 1 μg of pJ6Ωpuro (gift from J. Morgenstern) ). During transfection, 293 growth medium Was supplemented with 1.0 μg / ml tetracycline (Sigma). Transfect The washed cells were combined with 1.0 μg / ml tetracycline and 2 μg / ml 4 transfection into 293 growth medium supplemented with Seeded for 8 hours of selection. 72 independent clones for clone expansion Selected and screened for tetracycline-inducible VSV-G expression. To screen clones, each clone was treated at 30% confluence. Two 35 mm tissue culture dishes (Corning) were seeded simultaneously. The next day, one play Washed twice with 2 ml of 293 growth medium without tetracycline and The medium was changed to standard 293 medium supplemented with g / ml puromycin. 48 At time, cells are harvested for whole cellular protein and a pair of samples is 7.5% under reducing conditions. On a SDS-polyacrylamide gel. Semi-dry elect the gel Nitrocellulose (Schleicher & Schuell, O.L. 45 mm). Western blotting was performed using standard techniques . For the primary antibody, a murine monoclonal anti-VSV-G IgG (Sigma) Used at a dilution of 1: 800. For the secondary antibody, HRP-conjugated donkey anti-mouse Ig The GF (ab) 2 fragment (Pharmingen) was used at a dilution of 1: 10,000. Dupont  Chemiluminescence detection was performed using the NEN Renaissance kit. Tetra in growth medium There was no detectable VSV-G expression in the presence of cyclin and tetracycline was added to the growth medium. Based on the detection of inducible VSV-G expression in the absence of clin, Positive cell lines (such as H29) were selected.H29 gagpol cell line (293 GPG cell line).   H29 cells were treated with 1.0 μg / ml tetracycline and 2 μg / ml puro. The cells were grown in 293 growth medium (H29 medium) supplemented with mycin, 10 μg linearized with 2 μg of pSV2neo and ScaI by a chromium precipitation method PMD. Cotransfected with gagpol. Transfex During the incubation, H29 medium was supplemented with 1.0 μg / ml tetracycline. G Transfected H29 cells were treated with 1.0 μg / ml tetracycline and 0.1 μg / ml. Transfection was performed on H29 medium supplemented with 3 mg / ml G418 (Gibco). 48 hours after seeding. 69 independent clones And screened for reverse transcriptase activity (Goff et al., J. Virology, 3 8: 239-248 (1981)).   Of the 69 independent clones selected for clone expansion, 10 The clone has reverse transcriptase activity over the positive control (ie, Ψ Cre cells). Was. These ten clones are further characterized.H29 new gagpol cell line   H29 cells were treated with 1.0 μg / ml tetracycline and 2 μg / ml puro. The cells were grown in 293 growth medium (H29 medium) supplemented with mycin, 10 μg linearized with 2 μg of pSV2neo and ScaI by a chromium precipitation method PMD. Co-transfected using new gagpol. Trance During transfection, H29 medium was supplemented with 1.0 μg / ml tetracycline did. 1.0 μg / ml tetracycline was added to the transfected H29 cells. In H29 medium supplemented with G418 (Gibco) at 0.3 mg / ml. Seed was selected for 48 hours after injection. Independent clones And screened for reverse transcriptase activity (Goff et al., J. Virology (19). 81)).Consideration   Therefore, a derivative of pMD, pMDtet. G is VSV. G Tetra Provides cyclin-inducible expression. Inducible expression of VSV-G is: Transient assays and were shown in a stable cell line, H29. H29 is 293 thin Vesicles (Graham, F. et al., J. Gen. Virol., 36: 59-72 (1977)) and pBC. tTA (Tet transactivator), pMDtet. G and pJGΩ Selected after co-transfection with puro. H29 cells are derived from the parental 29 Inducible VSV-G only 5 times lower than transient VSV-G expression in 3 cells Is shown by Western blotting. H29 cells have an inducible V Twenty consecutive subcultures were repeated to show the expression of SV-G.Example 3 Construction of ΔU3 retrovirus cloning vector   The ΔU3nlsLacZ retroviral vector was constructed using MFG. SnlsLacZ (Berns et al., Human Gene Therapy, 6: 347-368 (1995); see FIG. 8). Of the U3 region of HCMV enhancer-promoter (nt-671 -2) (Bo shart, M., Cell, 41: 521-530 (1985)). ΔU3 nlsLacZ, the complete 5 'genomic flanking region and MFG. S All but 65 bp from the 3 'genomic flanking region from nlsLacZ Is removed.   The pMD plasmid was constructed as described in Example 1. ΔU3nlsLac For the construction of Z, a 701 bp fragment encoding the HCMV promoter was Using the pMD plasmid as a template, a 5 'HindIII site and a 3' A pair of primers to create, Was prepared by PCR. Digest the PCR product with HindIII and KasI Thus, a 677 bp fragment was obtained. MFG (Riviere, I. et al., PNAS, 92: 6733-6737 (1 995)), a 91 bp KasI-StyI was isolated from the 3 'LTR. 253b p StyI-EagI and a 4994 bp EagI-ScaI fragment were G. FIG. From SNlsLacZ (Berns et al., Human Gene Therapy, 6: 342-368 (1995)). The isolated, ΔU3nlsLacZ backbone is a 2.65k from pUC18 b is a HindIII-SmaI fragment.   For the construction of ΔU3Bam, the 561 bp fragment was replaced with ΔU3nlsLacZ. Is used as a template to create a 5 'KasI site and a 3' BamHI site. Immer, Was prepared by PCR. Digest the PCR product with KasI and BamHI A 389 bp fragment was obtained. The 389 bp KasI-BamHI fragment was converted to ΔU3 4466 bp BamHI-EagI from nlsLacZ and 695 bp Ligation with the EagI-KasI fragment.   FIG. 9A shows the structure of the ΔU3nlsLacZ vector, and FIG. 1 shows the structure of an am vector.Example 4 VSV-G plasmid by transient transfection of 293GPG cells Production of pseudotyped retrovirus   Plasmid pBC. tTA was converted to pUHD10-1 (Gossen, M. et al., Proc. Natl. . Acad. Sci. 89: 5547-551 (1992)) Substitution of the IL-2 sequence (bp 756-1439) with the offspring resulted in pBC12 / CM V / IL-2 (Cullen, BR, Cell, 46: 973-982 (1986)). FIG. Is pBC. 1 shows the structure of the tTA plasmid.   pMD, pMD. G, pMDtet, pMDtet. G and pMD. gag The pol construct as well as the 293GPG cell line were constructed as described in Example 1. .   Transient transfections with 293GPG cells were performed at 4-5 x 10⁶ Cells were plated overnight in a 60 mm dish seeded in 4 ml of 293GPG medium. Done. 4 μg of ΔU3nlsLacZ was added to 300 μl of Opti-MEM (Gib co BRL) and 25 μl L diluted in 300 μl Opti-MEM Incubate at room temperature for 30 minutes with ipofectamine (Gibco BRL). I did it. 2.4 ml of Opti-MEM was transferred to DNA-Lipofectamine. e, rinsed 30 minutes before transfection and added 2 ml Op The cells were placed on top of 293GPG cells whose medium had been changed to ti-MEM. Trance Seven to eight hours after transfection, 2 ml of 293 medium was added and the medium was Was replaced by Supernatants were collected at 72 hours and virus titers were determined as follows. .Assay of β-galactosidase activity and determination of virus titer   To stain cells for β-galactosidase activity, cells were washed with 1 mM After washing with phosphate buffered saline (PBS +) supplemented with gnesium, PBS + In 1% glutaraldehyde for 10 minutes at 37 ° C. (Lim, K. et al. Biotechniques, 7: 576-579 (1989)). The fixative is aspirated off and the cells are washed with PBS + 3.3 mM potassium ferricyanide (Sigma) in medium, 3.3 mM ferrocyanation With potassium (Sigma) and 0.2% X-gal (Molecular Probes) Incubated at 7 ° C. for 2 hours. Quantitative β-galactosidase activity is commercially available Was measured using a luminescence assay (Clontech). To measure the virus titer, NIH 3T3 cells were injected 1 × 1 per well in a 6-well culture dish 16 hours prior to infection. 0^FiveSerial of virus supernatant seeded with cells and containing 8 μg / ml polybrene (Sigma) Incubated with diluent for 24 hours. The virus titer is 20 1 mm^Two Number of cells with blue nuclei (β-galactosidase producing cells) per field of view × Factor occupying plate size, dilution of virus stock solution に お け る in tissue culture well It was determined as a target cell.VSV-G pseudotype by transient transfection of 293GPG cells Production of retroviruses   High transfection efficiency of 293 cells using 293GPG clone Utilization and pseudotyping with VSV-G by transient transfection Retrovirus was produced. Transient transfection of 293GPG cells For MFG. The U3 region of the 5 'LTR of nlsLacZ was Replacement with enhancer-promoter and MFG. 5 'and 3 of SnlsLacZ 'U3nlsLacZ retrovirus due to deletion of the genomic flanking region Constructs were made. MFG. ΔU3nls when compared to SnlsLacZ LacZ enables a 20-fold increase in expression in transient transfection did. The 293GPG clone was transformed with the ΔU3nlsLacZ construct at an average efficiency of 40%. Transiently transfected with lipofectamine using a build. Trance The virus supernatant was collected between 24 and 72 hours post-fection for 48 hours, and Tetracycline was removed from the growth medium. 1-3x10⁶i. u. / Ml range Of the virus was transiently achieved.Example 5 Effect of 5 'untranslated sequence on cDNA expression and virus titer   Between previously used vector and ΔU3 retroviral cloning vector There are some differences. First, the ΔU3 vector is the U3 of the 5 'LTR. The region is a complete human CMV enhancer-promoter (Boshart et al., Cell, 41:52). 1-530 (1985)) to obtain a 293-derived cell line (293G). PG cells) have been specifically modified for high transient expression. Second , ΔU3 vector is derived from MGF, an established high titer and high expression vector You. Finally, what was the vector used for previous retroviral expression cloning? Differently, the effect of 5 'untranslated sequence on cDNA expression and cDNA virus titer Tested. This is due to the process of constructing the cDNA library (Seed, B. and Aruffo, S., PNAS, 84: 3365-3369 (1987)). Important because it includes the 5 'untranslated sequence up to the base pair. Therefore, retro ui The Lus cloning vector should be used to avoid introducing bias into the library. It must be possible to apply these additional arrays.   The effect of the 5 'untranslated sequence on cDNA expression and virus titer Insert the lacZ gene with 0-165 base pairs in the sequence into the ΔU3Bam vector Tested by: The following shows 0 base pairs of the untranslated sequence (ie, modified (U3lacZ without BamHI cloning site) The data is:   Based on these results, the ΔU3Bam vector was based on uncloned MGF. Moderately reduced expression or viral titer compared to retroviral vectors Applying up to 165 bp of 5 'untranslated sequence in the cDNA insert with only a few It is shown that can be. This data indicates that the ΔU3Bam vector Efficient packaging of input and efficient cDNA to target cells (call) Support the possibility of promoting effective communication.Equivalent   Those skilled in the art will recognize, by mere routine experimentation, the invention described herein. Many equivalents to a particular embodiment can be recognized or confirmed. Would. Such equivalents are included within the scope of the invention as set forth in the following claims. It is what is done.

[Procedural Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] December 9, 1997 (1997.12.9) [Contents of Amendment] Claims 1. A stable pseudotyped retroviral packaging cell line that produces a helper-free recombinant pseudotyped retrovirus, comprising: a) a retroviral gagpol gene that produces a retroviral core viral particle; and b) a retroviral core viral particle. A protein encoding a pseudotyped envelope protein and under the control of an inducible operator system (where b) comprises a stable pseudotyped retrovirus packaging that produces a helper-free recombinant pseudotyped retrovirus. A packaging cell comprising one or more non-retroviral expression constructs that governs expression of a retroviral core virion that produces a cell line, providing a pseudotyped envelope protein for the retroviral core virion. Strain. 2. A stable pseudotyped retroviral packaging cell line producing a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range derived from human, comprising: a) a retrovirus producing a retroviral core virus particle gagpol gene; and b) a gene encoding a pseudotyped envelope protein for the retroviral core and under the control of an inducible operator system, wherein the vesicular stomatitis virus G glycoprotein is a helper with a pan-affinity host range. Governs the expression of a pseudotyped retrovirus, which provides a stable pseudotyped retrovirus packaging cell line that produces a free recombinant pseudotyped retrovirus, providing a pseudotyped envelope protein for the retroviral core virion Packaging cell lines comprising the more non-retroviral expression construct. 3. 3. The stable pseudotyped retroviral packaging cell line of claim 2, wherein the envelope protein of b) is vesicular stomatitis virus G glycoprotein. 4. 4. The packaging cell line of claim 3, wherein the non-retroviral expression construct comprises a human cytomegalovirus immediate early promoter. 5. 4. The packaging cell line of claim 3, wherein the inducible operator system for vesicular stomatitis virus G glycoprotein expression is a tet operator system. 6. The packaging cell line according to claim 3, wherein the retrovirus gagpol gene is mutated. 7. 4. The packaging cell line of claim 3, wherein the cell is a human cell expressing VSV-G under the control of an inducible tet operator, an H29 gagpol cell comprising tTA and gagpol. 8. 7. The packaging cell line of claim 6, wherein the cell is a human cell expressing VSV-G under the control of an inducible tet operator, a tTA, and an H29 new gagpol cell comprising a mutated gagpol gene. . 9. A stable pseudotyped retrovirus packaging cell line producing a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range, comprising: a) a retroviral gagpol gene producing retroviral core virus particles; and b. ) Vesicular stomatitis virus G glycoprotein under the control of an inducible tet operator system, wherein the vesicular stomatitis virus G glycoprotein produces a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range Providing one or more cytomegalovirus expression constructs that govern expression of a retrovirus core virion, which results in a stable pseudotyped retroviral packaging cell line. Packaging cell line. 10. 10. The packaging cell line of claim 9, wherein the cell is a human cell expressing VSV-G under the control of an inducible tet operator, an H29 gagpol cell comprising tTA and gagpol. 11. A stable pseudotyped retrovirus packaging cell line producing a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range, comprising: a) a mutated retroviral gagpol gene; and b) an inducible tet operator. Vesicular stomatitis virus G glycoprotein under system control, wherein the vesicular stomatitis virus G glycoprotein is a helper-free recombinant pseudotype having a pan-affinity host range from a stable pseudotype retrovirus packaging cell line. To provide a pseudotyped envelope protein that interacts with the retroviral gagpol protein expressed from the mutated gagpol gene to produce the retrovirus of the present invention. Packaging cell lines that were formed by. 12. 12. The packaging cell line of claim 11, wherein the cell is a human cell expressing VSV-G under the control of an inducible tet operator, a tTA, and an H29 new gagpol cell comprising a mutated gagpol gene. 13. a) a retroviral gagpol gene that produces a retroviral core virion, and b) a protein that provides a pseudotyped envelope for the retroviral core virion and is under the control of an inducible operator system. The protein of b) provides a pseudotyped envelope protein for the retroviral core virion, which results in a stable pseudotyped retroviral packaging cell line that produces a helper-free recombinant pseudotyped retrovirus). A stable pseudotyped retroviral packaging cell line producing a helper-free recombinant pseudotyped retrovirus, comprising transfecting a mammalian cell with one or more predominant non-retroviral expression constructs Manufacturing method. 14． a) the retroviral gagpol gene that produces the retroviral core virion, and b) the vesicular stomatitis virus G glycoprotein under the control of an inducible operator system, wherein the vesicular stomatitis virus G glycoprotein is a pancreatic stomatitis virus G glycoprotein. Which provides a pseudotyped envelope protein for the retroviral core virion, resulting in a stable pseudotyped retroviral packaging cell line producing a helper-free recombinant pseudotyped retrovirus having an affinity host range. Stable pseudotyped retroviral packaging producing a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range, comprising transfecting mammalian cells with one or more predominant non-retroviral expression constructs A method for manufacturing a 胞株. 15. 15. The method of claim 14, wherein the non-retroviral expression construct is a cytomegalovirus construct. 16. 15. The method according to claim 14, wherein the inducible operator system for the expression of vesicular stomatitis virus G glycoprotein is a tet operator system. 17． 15. The method of claim 14, wherein the mammalian cells are human cells that express VSV-G under the control of an inducible tet operator, and H29 cells that comprise tTA. 18. The method according to claim 14, wherein the retrovirus gagpol gene is mutated. 19. 19. The method of claim 18, wherein the mutated gagpol gene comprises the new 5 'gagpol and new 3' gagpol nucleotide sequences, SEQ ID NOs: 7 and 8. 20. a) transfecting a mammalian cell with a first non-retroviral construct encoding a tet transactivator and a second non-retroviral construct encoding a vesicular stomatitis virus G glycoprotein under the control of the tet transactivator B) screening the cells of a) for tetracycline-inducible VSV-G expression that is not detected in the presence of tetracycline and is detected in the absence of tetracycline; b) retrovirus; transfecting the cells of b) with a third non-retroviral construct encoding the gagpol protein; and d) screening the cells of c) for production of the retrovirus, wherein d producing the retrovirus. ) Transfer (A) is a stable pseudotyped retrovirus packaging cell that produces a helper-free recombinant pseudotyped retrovirus having a pan-affinity host range). A method for producing a stable pseudotype retrovirus packaging cell line that produces a recombinant pseudotype retrovirus. 21. 20. The method of claim 20, wherein the second and third constructs are cytomegalovirus constructs. 22. 21. The method of claim 20, wherein the mammalian cells are human cells that express VSV-G under the control of an inducible tet operator, and H29 cells that comprise tTA. 23. 21. The method according to claim 20, wherein the retroviral gagpol protein is mutated. 24. 24. The method of claim 23, wherein the mutated gagpol protein comprises the new 5 'gag and new 3' pol proteins encoded by SEQ ID NOs: 7 and 8. 25. Human cells expressing VSV-G under the control of an inducible tet operator, and an H29 cell line comprising tTA. 26. H29 gagpol cell line, comprising human cells expressing VSV-G under the control of an inducible tet operator, tTA, and gagpol. 27. H29 new gagpol cell line comprising human cells expressing VSV-G under the control of an inducible tet operator, tTA, and a mutated gagpol gene. 28. PMD comprising the vector of FIG. 29. PMDtet comprising the vector of FIG. 30. PMDtet. Containing the vector of FIG. G. 31. PMD. Containing the vector of FIG. gagpol. 32. PMD. Containing the vector of FIG. new gagpol. 33. a) two retroviral LTRs; b) a cloning site for insertion of a cDNA; and c) a retroviral vector for construction of a cDNA library for expression in mammalian cells, comprising a cytomegalovirus promoter. . 34. 34. The retroviral vector according to claim 33, wherein the retroviral LTR is Moloney murine leukemia virus LTR. 35. 35. The retroviral vector according to claim 34, wherein one of said LTRs is a modified Moloney murine leukemia virus LTR wherein the U3 region of Moloney murine leukemia virus LTR is replaced with a human cytomegalovirus promoter.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), CA, JP, US (72) Inventors Auri, Daniel S.             United States Missouri 63124 Sen             Truis, Greenbrier Street             To 8 (72) Inventor Sedrain, Michael             United States New York 10128             New York, East 89 Tea H               Street 401 (72) Inventor Mulligan, Richard Sea.             United States California 94705               Berkeley, Bell Rose Avenue             2337 (72) Inventor Shafar, Gene E.             United States Missouri 63124 Sen             Truis, Greenbrier Street             To 8 [Continuation of summary] Special cells and constructs (ie packaging Element) (H29 cells and pMD, pMDtet, pMDte t. G, PMD. gagpol, pMD. new ga gpol constructs). The present invention has two retro Cloni for insertion of viral long terminal repeats, cDNA Site and the cytomegalovirus promoter CDNA library for expression in mammalian cells And a retrovirus vector for the production of Also The present invention contains the retroviral vector of the present invention. CDNA library for expression in mammalian cells I do. The present invention also provides expression clones in mammalian cells. The method of The present invention also relates to mammalian cells. CDNA cloning method. Again The expression of cDNA by complementation in mammalian cells Of genes responsible for mutated phenotypes using The present invention relates to a method for identifying a defect.

Claims (1)

[Claims] 1. An inexpensive, capable of producing a helper-free recombinant pseudotyped retrovirus A constant pseudo-type retroviral packaging cell line, a) Retrovirus gagpol, which produces a retrovirus core virus particle A gene; and b) Pseudo-type envelope for retroviral core virus particles Genes under the control of an encoded and inducible operator system (Here, the protein (b) is a helper-free recombinant pseudotype retrovirus. Stable pseudotyped retroviral packaging cells producing virus A pseudotyped envelope for the retroviral core virus particle that yields the strain Provides rope protein) A package comprising one or more non-retroviral expression constructs that direct the expression of Zing cell line. 2. Helper-free recombinant pseudotyped retrovirus with pan-affinity host range A stable pseudo-type retrovirus packaging cell capable of producing ills. Cell strain, a) Retrovirus gagpol, which produces a retrovirus core virus particle A gene; and b) Vesicular stomatitis virus G glycoprotein under control of an inducible operator system (Here, the vesicular stomatitis virus G glycoprotein has a pan-affinity host range. A stable pseudotype that produces a purfic recombinant pseudotyped retrovirus Retroviral coaus yielding a type of retroviral packaging cell line Provides pseudo-type envelope protein for ills particles) A package comprising one or more non-retroviral expression constructs that direct the expression of Zing cell line. 3. Non-retroviral expression construct is a human cytomegalovirus immediate early promoter The packaging cell line according to claim 2, comprising a protein. 4. Inducible operator system for expression of vesicular stomatitis virus G glycoprotein The packaging cell line of claim 2, wherein is a tet operator system. 5. The package according to claim 2, wherein the retrovirus gagpol gene is mutated. Ging cell line. 6. 3. The packaging cell of claim 2, wherein the cells are H29 gagpol cells. Spore strain. 7. The package according to claim 5, wherein the cells are H29 new gagpol cells. Zing cell line. 8. Helper-free recombinant pseudotyped retrovirus with pan-affinity host range A stable pseudo-type retrovirus packaging cell capable of producing ills. Cell strain, a) Retrovirus gagpol, which produces a retrovirus core virus particle A gene; and b) Vesicular stomatitis virus G glycoprotein under the control of an inducible tet operator system quality (Here, the vesicular stomatitis virus G glycoprotein has a pan-affinity host range. A stable pseudotype that produces a purfic recombinant pseudotyped retrovirus Retroviral coaus yielding a type of retroviral packaging cell line Provides pseudo-type envelope protein for ills particles) Comprising one or more cytomegalovirus expression constructs that govern expression of Caging cell line. 9. 9. The packaging cell of claim 8, wherein the cells are H29 gagpol cells. Spore strain. 10. Helper-free recombinant pseudotype retro with pan-affinity host range Stable pseudo-type retrovirus packaging capable of producing virus A cell line, a) a mutated retroviral gagpol gene; and b) Vesicular stomatitis virus G glycoprotein under the control of an inducible tet operator system quality (Here, the vesicular stomatitis virus G glycoprotein is a stable pseudo-type Helper-free with pan-affinity host range from rovirus packaging cell lines In order to produce a recombinant pseudotyped retrovirus, the mutated gagpo that interacts with the retroviral gagpol protein expressed from the l gene Provides an id-type envelope protein) Comprising one or more cytomegalovirus expression constructs that govern expression of Caging cell line. 11. The package according to claim 10, wherein the cells are H29 new gagpol cells. Caging cell line. 12. a) Retroviral gag producing retroviral core viral particles ol gene, and b) Pseudo-type envelope for retroviral core virus particles Provided and inducible proteins under the control of an operator system (Here, the protein (b) is a helper-free recombinant pseudotype retrovirus. Stable pseudotyped retroviral packaging cells producing virus A pseudotyped envelope for the retroviral core virus particle that yields the strain Provides rope protein) Transfecting mammalian cells with one or more non-retroviral expression constructs that govern expression of Helper-free recombinant pseudo-type retro Stable pseudo-type retrovirus packaging cells capable of producing ills How to make a strain. 13. a) Retroviral gag producing retroviral core viral particles ol gene, and b) Vesicular stomatitis virus G glycoprotein under control of an inducible operator system (Here, the vesicular stomatitis virus G glycoprotein has a pan-affinity host range. A stable pseudotype that produces a purfic recombinant pseudotyped retrovirus Retroviral coaus yielding a type of retroviral packaging cell line Provides pseudo-type envelope protein for ills particles) Transfecting mammalian cells with one or more non-retroviral expression constructs that govern expression of A helper-free recombinant plasmid with a pan-affinity host range, comprising the step of transfecting Stable pseudo-type retrovirus capable of producing pseudo-type retrovirus A method for producing a ruth packaging cell line. 14． Claims wherein the non-retroviral expression construct is a cytomegalovirus construct Item 14. The method according to Item 13. 15. Inducible Operator for Expression of Vesicular Stomatitis Virus G Glycoprotein 14. The method according to claim 13, wherein the system is a tet operator system. 16. 14. The method according to claim 13, wherein the mammalian cells are H29 cells. 17． 14. The method according to claim 13, wherein the retrovirus gagpol gene is mutated. Law. 18. The mutated gagpol gene is new 5 'gagpol and new 18. The method of claim 17, which is a 3 'gagpol nucleotide sequence. 19. a) a first non-retroviral construct encoding a tet operator and Second non-retroviral construct encoding vesicular stomatitis virus G glycoprotein Transfecting a mammalian cell at b) VSV-G was not detected in the presence of tetracycline, Is associated with tetracycline-inducible VSV-G expression detected in the absence of And screening the cells of a); c) a third non-retroviral construct encoding the retroviral gagpol protein Transfecting the cells of b) with a building; d) screening the cells of c) for retrovirus production (Where the retrovirus producing d) transfected cells were Helper-free recombinant pseudotyped retrovirus with compatible host range It is a stable pseudo-type retrovirus packaging cell that can be produced.) A helper-free recombinant pseudotype reto having a pan-affinity host range comprising Stable pseudo-type retrovirus packaging capable of producing rovirus How to make cell lines. 20. 2. The method according to claim 1, wherein the second and third constructs are cytomegalovirus constructs. 9. The method according to 9. 21. 20. The method according to claim 19, wherein the mammalian cells are H29 cells. 22. The method according to claim 19, wherein the retrovirus gagpol protein is mutated. . 23. The mutated gagpol protein was identified as new 5 'gag and new 3' p 20. The method according to claim 19, which is an ol protein. 24. H29 cell line. 25. H29 gagpol cell line. 26. H29 new gagpol cell line. 27. pMD. 28. pMDtet. 29. pMDtet. G. 30. pMD. gagpol. 31. pMD. new gagpol. 32. a) two retroviral LTRs; b) a cloning site for insertion of the cDNA; and c) Cytomegalovirus promoter For production of a cDNA library for expression in mammalian cells, comprising Retrovirus vector. 33. The retroviral LTR is Moloney murine leukemia virus LTR. 33. The retroviral vector according to claim 32. 34. One of the LTRs is the Moloney murine leukemia virus LTR U3 region. Modified Moloney murine replaced with the cytomegalovirus promoter 34. The retroviral vector according to claim 33, which is a myeloid leukemia virus LTR. 35. The library is a) two retroviral LTRs; b) cDNA; and c) Cytomegalovirus promoter (Here, the cDNA is located between the retroviral LTRs, Operably linked to the Ruth promoter) C for expression in mammalian cells, comprising a retroviral vector containing DNA expression library. 36. The retrovirus LTR is Moloney murine leukemia virus LTR; One of the LTRs is that the Moloney murine leukemia virus LTR U3 region is a human site. Modified Moloney murine leukemia replaced by megalovirus promoter The cDNA expression library according to claim 35, which is a viral LTR. 37. a) Two retroviral LTRs, cDNA and cytomegalovirus Retroviral vector containing promoter (Here, the cDNA is located between the retroviral LTRs, Operably linked to the Ruth promoter) Introducing into a mammalian cell a cDNA expression library comprising Bini b) c prepared in a) under conditions suitable for expression of the cDNA expression library Maintaining mammalian cells containing a DNA expression library Thereby producing a cDNA expression library in said mammalian cells. A method for producing a cDNA expression library in a mammalian cell. 38. Uses pseudotype retrovirus produced in packaging cell line 38. The method of claim 37, wherein the cDNA expression library is introduced into a cell. 39. a) Two retroviral LTRs, cDNA and cytomegalovirus Retroviral vector containing promoter (Here, the cDNA is located between the retroviral LTRs, Operably linked to the Ruth promoter) A cDNA expression library comprising a pseudotyped retrovirus Transfer to a particle-producing packaging cell line, thereby allowing expression libraries Producing a packaging cell line comprising: b) a pseudo-type containing RNA transcribed from said cDNA expression library Under conditions suitable for the production of retroviral particles, a package containing the expression library Maintaining a ging cell line; c) Infecting mammalian cells with the pseudotype retrovirus of b) Producing a cDNA expression library in mammalian cells. A method for preparing a cDNA expression library in a mammalian cell. 40. The packaging cell lines are 293GPG cells, BOSC23 cells, PA3 17 cells, Kat cell line, GP + E-86 cell line, GP + EAM12 cell line and 40. The method of claim 39, wherein the method is selected from the group consisting of: 41. a) 1) two Moloney murine leukemia virus LTRs;             2) cDNA; and             3) Cytomegalovirus promoter (Here, the cDNA is located between the Moloney murine leukemia virus LTRs, (It is operably linked to the Itomegalovirus promoter.) CDNA expression library containing a retroviral vector containing Making; b) using the cDNA expression library to produce pseudotyped retroviruses Introducing into a mammalian packaging cell line; and c) Production of RNA transcript from cDNA in said cDNA expression library and Conditions suitable for producing pseudotyped retroviral particles containing RNA transcripts Maintaining the mammalian packaging cell line in A broad host range containing RNA transcribed from a cDNA expression library A method for producing pseudotype retrovirus particles. 42. Pseudo-type retroviral particles are pseudotyped with VSV-G 42. The method of claim 41, wherein the retroviral particle is a modified retroviral particle. 43. 43. A broad host range pseudotype produced by the method of claim 42. Retroviral particles. 44. Retrovirus for Creating cDNA Library for Expression in Mammalian Cells The retroviral vector comprising RNA transcribed from an ills vector. -Two retrovirus LTRs; cloning site for insertion of cDNA And a broad host range promoter comprising the cytomegalovirus promoter. Pseudo-type retroviral particles. 45. 45. The pseudotype according to claim 44, which is of the VSV-G retrovirus pseudotype. Id-type retroviral particles.