WO1998029533A1 - Vectors and methods for the mutagenesis of mammalian genes - Google Patents
Vectors and methods for the mutagenesis of mammalian genes Download PDFInfo
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- WO1998029533A1 WO1998029533A1 PCT/US1997/023956 US9723956W WO9829533A1 WO 1998029533 A1 WO1998029533 A1 WO 1998029533A1 US 9723956 W US9723956 W US 9723956W WO 9829533 A1 WO9829533 A1 WO 9829533A1
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- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
Definitions
- the gene trap approach is limited to genes expressed in ES cells, although variations of the method have been developed for targeting specific subclasses of genes expressed in early embryonic stages (Wurst et al. Genetics 139:889-899 (1995); Skarnes et al, Proc. Natl. Acad. Sci. USA 92:6592-6596 (1995); and Forrester et al, Proc. Natl. Acad. Sci. USA 93: 1677-1682 (1996)). And the chemical/radiation induced mutagenesis technique is generally limited to genes that can result in dominant phenotypes when mutated. None of these approaches, as currently exploited, may be readily streamlined or automated, nor can they be readily adapted to carry out saturated mutagenesis of the mouse genome.
- the invention includes a cell containing a retroviral vector of the invention; a transgenic non-human mammal (for example, a mouse) which includes a retroviral vector of the invention; a library (that is, having at least 100 members) of mutagenized mammalian genes produced by the methods of the invention; and cells (for example, stem cells) which include a library of mutagenized mammalian genes produced by the methods of the invention.
- the invention features a method for identifying a cell (for example, a stem cell) which includes a retroviral vector, the method involving: (a) introducing into a mammalian cell population a retroviral vector, the vector including a splice acceptor sequence, a transcription termination sequence, retroviral packaging and integration sequences, and a constitutively expressed detectable marker gene, the introducing step being carried out under conditions which allow the vector to integrate into the genomes of the cells; and (b) identifying the cell which includes the retroviral vector by detecting expression of the marker gene.
- the marker gene is a green fluorescent protein; and the green fluorescent protein has increased cellular fluorescence relative to the wild- type green fluorescent protein.
- the invention features a method of conditionally ablating a cell lineage, the method involving: (a) providing a first transgenic non- human mammal which includes an activator protein expressed only in the cell lineage; (b) providing a second transgenic non-human mammal which includes a nucleic acid sequence encoding a cell ablation factor, the nucleic acid sequence being under the control of the activator protein and the activator protein being capable of binding to and regulating the nucleic acid sequence only upon induction; (c) mating the first and the second transgenic mammals to produce offspring in which the cell ablation factor is expressed under the control of the activator protein, the cell ablation factor being capable of destroying cells in which it is expressed; and (d) inducing binding and regulation by the activator protein.
- the activator protein is introduced into the transgenic non-human mammal on a retroviral vector that includes a splice acceptor sequence, a transcription termination sequence, and retroviral packaging and integration sequences;
- the activator protein is a tetracycline repressor fused to VP16 and the nucleic acid sequence encoding a cell ablation factor is operably linked to a tetracycline operator;
- the cell ablation factor is chosen from the group consisting of a toxin, a thymidine kinase, or an apoptotic protein; the conditional induction occurs by administration of tetracycline or a tetracycline derivative to the transgenic mammal; and the mammal is a mouse.
- the invention features a method for conditional ectopic expression of a gene of interest, the method involving: (a) providing a first transgenic non-human mammal which includes an activator protein expressed under the control of the promoter of an endogenous gene of the mammal; (b) providing a second transgenic non-human mammal which includes a nucleic acid sequence encoding the gene of interest, the nucleic acid sequence being under the control of the activator protein and the activator protein being capable of binding to and regulating the nucleic acid sequence only upon induction; (c) mating the first and the second transgenic mammals to produce offspring in which the gene of interest is expressed under the control of the activator protein; and (d) inducing expression of the activator protein.
- the invention features a method of generating a non- human transgenic mammal having a conditional malignancy, the method involving: (a) providing a first transgenic non-human mammal which includes an activator protein expressed under the control of the promoter of an endogenous gene of the mammal; (b) providing a second transgenic non-human mammal which includes a nucleic acid sequence encoding a neoplastic factor, the nucleic acid sequence being under the control of the activator protein and the activator protein being capable of binding to and regulating the nucleic acid sequence only upon induction; (c) mating the first and the second transgenic mammals to produce offspring in which the neoplastic factor is expressed under the control of the activator protein, the neoplastic factor being capable of promoting the development of the malignancy; and (d) inducing binding and regulation by the activator protein.
- the activator protein is introduced into the transgenic non-human mammal on a retroviral vector that includes a splice acceptor sequence, a transcription termination sequence, and retroviral packaging and integration sequences;
- the activator protein is a tetracycline repressor fused to VP16 and the nucleic acid sequence encoding a cell ablation factor is operably linked to a tetracycline operator;
- the neoplastic factor is an oncogene; the induction occurs by administration of tetracycline or a tetracycline derivative to the transgenic mammal; and the mammal is a mouse.
- the invention also features a cell line derived from one of these transgenic non- human mammals, as well as transgenic mosaic non-human mammals generated by the methods of the invention and uses therefor.
- the present invention provides a number of advantages. For example, it combines versatile retroviral vectors for ES cell mutagenesis with powerful detection methods for rapid identification of mutant cells of interest.
- the method permits mutagenesis in a large number of mammalian genes, in a short period of time, and at a significant reduction in cost.
- the method may be readily streamlined, and many genes may be processed in parallel. Considering that every gene is a potential mutagenesis target, the proposed approach facilitates the generation of extensive libraries of mutated mammalian genes, as well as libraries of pluripotent stem cells carrying those gene mutations.
- FIGURE 3 is a schematic representation of the MAGEKO process of insertional mutagenesis in an exon sequence.
- Retroviruses are RNA viruses which replicate through a DNA intermediate and which include as an obligatory step of their life cycle integration of the proviral DNA into the host chromosome (Varmus and Brown, Retroviruses. In Mobile DNA (ed. Berg, D. E. and M. M. Howe), pp. 53-108. American Society for Microbiology, Washington, D.C. (1989)). Following integration, the provirus is maintained as a stable genetic element in the infected cell and its progeny. Most or possibly all regions of the host genome are accessible to retroviral integration (Withers- Ward et al. Genes Dev. 8: 1473-1487 (1994)), and the above properties make retroviruses invaluable as both potent mutagens and chromosomal markers.
- V. V, or VDErs is the recognition sequence of the VDE DNA endonuclease from Saccharomyces cerevisiae (Bremer et al. Nucleic Acids Res. 20:5484 (1992)). This sequence provides a unique chromosomal marker. Other chromosomal markers may also be utilized for this purpose.
- a preferable consensus splice acceptor is derived from the Adenovirus major late transcript (Robberson et al, Mol. Cell. Biol.
- P. P is the constitutively expressed mouse phosphoglycerate kinase- 1 (PGK) promoter (Adra et al. Gene 60:65-74 (1987)). This promoter is required for the expression of GFO (described below). Other constitutive mammalian promoters may be used in place of the PGK sequence.
- PGK mouse phosphoglycerate kinase- 1
- the vectors of the invention possess a number of unique properties, making them useful for various types of gene disruption methods and types of analyses. Examples of these unique properties and uses now follow.
- the retroviral vectors are highly mutagenic.
- One significant advantage provided by the present retroviral vectors is the fact that these vectors are highly mutagenic. This property arises, at least in part, because the vectors contain a combination of a consensus splice acceptor and transcriptional termination sequences.
- the splice acceptor has been previously described (Gossler et al. Science 244:463-465 (1989); Friedrich and Soriano, Genes Dev. 5:1513-1523 (1991); Skarnes et al. Genes Dev.
- Insertion of the retroviruses into a gene of interest leads to gene inactivation which is independent of the site of integration.
- Normal transcription and subsequent translation of gene X (Fig. 2) are disrupted, whether or not the retroviral insertion has occurred in an exon sequence (Fig. 3) or an intron sequence (Fig. 4).
- This advantage is quite important.
- gene disruption is generally expected following integration of standard retroviruses into exons, the outcome of retroviral integration into introns is less predictable, and only a small fraction of retroviral insertions have been found to be associated with recessive phenotypes in the mouse (Jaenisch, Science 240:1468-1474 (1988)).
- the combination of a splice acceptor sequence and a transcriptional terminator is an important feature of the present invention, rendering the presently described vectors highly mutagenic even when integrated at intron locations.
- the MAGEKO method allows rapid identification of infected cells.
- the invention allows rapid identification of infected cells.
- the vectors of the invention include a marker which facilitates the identification of vector-containing cells.
- the vectors carry a GFP mutant with increased cellular fluorescence linked to the PGK promoter. This marker allows for the identification of infected cells hours after infection, thus enabling the rapid sorting of transduced cells, for example, by FACS analysis. This is an important element for the generation of LOKs.
- the marker may be any reporter of gene expression.
- reporter include, without limitation, the bacterial lacZ gene (An et al, Mol. Cell. Biol. 2: 1628- 1632 (1982)), green fluorescent protein, wavelength variations of green fluorescent protein (Heim et al, Proc. Natl. Acad. Sci. USA 91:12501-12504)), luciferase (de Wet et al, Mol. Cell. Biol. 7:725-737 (1987)), and chloramphenicol acetyltransferase
- mice are mated with mice carrying conditionally produced "cell ablation factors" which are themselves synthesized only in the presence of both rtTA and tetracycline derivatives.
- Offspring containing both transgenes are subjected to cell ablation studies following administration of tetracycline derivatives and resultant destruction of cells expressing the gene with the retroviral insertion. Examination of these offspring provides a functional characterization of the ablated cell lineage.
- Conditionally produced "cell ablation factors" useful in the invention include, but are not limited to, wild-type and mutant toxins (Borrelli et al. Nature 339:538-540 (1989); Frankel et al, Mol. Cell. Biol.
- mosaic mice are generated from homozygotic mutant ES cells in the gene of interest with mutant ES cells containing the identical proviral insertion in only one of the two alleles of the same gene.
- the heterozygotic cells (derived from animals generated as described above for the conditional ablation technique) also contain conditionally produced "cell ablation factors.” These factors are synthesized only in the presence of both rtTA and tetracycline derivatives, and rtTA, in turn, is produced only in cells expressing the gene with the retroviral insertion (Figs. 3 and 4).
- conditional tissue-specific gene inactivation may be employed.
- a gene of interest is inactivated, when desired, in the cells in which it is expressed.
- This general technique has been previously used to assign gene functions through the use of tissue-specific gene targeting (Gu et al. Science 265:103-106 (1994); Kuhn et al. Science 269:1427-1429 (1995); and Rajewsky et al, J. Clin. Invest. 96:600-603 (1996)).
- Conditional tissue-specific gene inactivation is accomplished through a binary transgenic mouse system, similar in principle to the one described above for conditional ablation of cell lineages.
- the mating partner carrying the "activator” is derived from heterozygotic mutant ES cells containing a retroviral insertion in one of the two alleles of the gene to be subjected to the conditional tissue-specific inactivation.
- This mouse produces rtTA only in cells synthesizing the target gene (Figs. 3 and 4).
- the other mating partner i.e., the one with the silent "weapon," carries a conditionally expressed ribozyme and a conditionally expressed recombinase.
- Ribozymes are molecules capable of catalyzing sequence specific cleavage of targeted RNAs (Airman, Proc. Natl. Acad. Sci. USA 90: 10898-10900 (1993)).
- the ribozyme is preferably expressed using an RNA polymerase III (Pol III) dependent promoter, such as the U6 small nuclear RNA promoter (Das et al, EMBO J. 7:503-512 (1988)).
- the Pol III promoter synthesizes the appropriate ribozyme only in the presence of rtTA and tetracycline derivatives.
- the constitutive Pol III promoter is preferably separated by transcription terminators from the ribozyme sequences.
- Flp or CRE is produced in cells expressing the target gene when tetracycline derivatives are administered to the animal.
- Production of Flp or CRE leads to recombinational excision of the termination sequences and synthesis of the ribozyme in those cells.
- the target gene is subjected to ribozyme action, and the phenotype of this conditional tissue- specific gene inactivation event is amenable to analysis.
- the oncogenic state is not constitutive, but is conditional; the neoplastic transformation of a normal mouse tissue is initiated only in the presence of tetracycline derivatives, making the system more amenable to analysis.
- Animals generated by this method provide information about the types of oncogenes which play roles in particular cell types, and may also be used as animal models to screen anti-cancer therapies.
- transduced cells expressing the visual marker are selected by FACS analysis, and the cells are distributed in multi-well plates.
- the contents of combinations of wells are then pooled, subsequent to duplicate formation and storage of the replica, and appropriate matrices are generated to facilitate assignment of a specific cell to a particular well.
- Several proposed and established pooling strategies are available for the generation of the desired matrix to screen the LOK (see, for example, Zwaal et al, Proc. Natl. Acad. Sci. USA 90:7431-7435 (1993); Evans and Lewis, Proc. Natl. Acad. Sci. USA 86:5030-5034 (1989); Green and Olson, Proc. Natl.
- Genomic DNA fragments, once amplified, are transferred to hybridization supports, generating an ordered array of genomic DNA flanking the provirus. Labelled DNA from a gene of interest is then hybridized to the pooled genomic DNA, and a positive signal leads to the rapid identification of the desired ES cell clone.
- oligonucleotides of a specific length are synthesized in a high density array (such as an Affymetrix chip (see, for example, Lipshutz et al, BioTechniques 19:442-447 (1995)) and hybridized to the amplified DNA from ES cells.
- the POP technique is based on generating sequence information for an unknown region of nucleic acid (i.e., the genomic DNA), which is linked to a known sequence (i.e., a portion of the retroviral vector). Because retroviral integration is precise and results in the integration of a viral LTR within the genomic DNA, the LTR sequence is a preferred sequence for designing oligonucleotide probes.
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JP53024298A JP2001507577A (en) | 1996-12-31 | 1997-12-31 | Vectors and methods for introducing mutations into mammalian genes |
AU58079/98A AU736267B2 (en) | 1996-12-31 | 1997-12-31 | Vectors and methods for the mutagenesis of mammalian genes |
CA002276529A CA2276529A1 (en) | 1996-12-31 | 1997-12-31 | Vectors and methods for the mutagenesis of mammalian genes |
EP97954255A EP0951533A4 (en) | 1996-12-31 | 1997-12-31 | Vectors and methods for the mutagenesis of mammalian genes |
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US20080260744A1 (en) * | 2002-09-09 | 2008-10-23 | Omeros Corporation | G protein coupled receptors and uses thereof |
US9453251B2 (en) | 2002-10-08 | 2016-09-27 | Pfenex Inc. | Expression of mammalian proteins in Pseudomonas fluorescens |
US7205148B2 (en) * | 2003-06-11 | 2007-04-17 | Regeneron Pharmaceuticals, Inc. | Genome mutation by intron insertion into an embryonic stem cell genome |
GB0325284D0 (en) * | 2003-10-29 | 2003-12-03 | Forinnova As | Methods of identifying active genes |
AU2005269527B2 (en) | 2004-07-26 | 2011-12-01 | Pfenex Inc. | Process for improved protein expression by strain engineering |
CA2909775A1 (en) | 2005-08-09 | 2007-03-29 | Revivicor, Inc. | Transgenic ungulates expressing ctla4-ig and uses thereof |
JP5301424B2 (en) * | 2006-03-15 | 2013-09-25 | ブライアン アール. ソーパー, | Methods for screening and mapping phenotypic and genotypic variations in cells |
US20070292842A1 (en) * | 2006-06-14 | 2007-12-20 | Fulmer-Smentek Stephanie B | Detection of viral or viral vector integration sites in genomic DNA |
US9580719B2 (en) | 2007-04-27 | 2017-02-28 | Pfenex, Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
CA2685326A1 (en) | 2007-04-27 | 2008-11-06 | Dow Global Technologies Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
JP5871304B2 (en) * | 2011-09-06 | 2016-03-01 | 大学共同利用機関法人自然科学研究機構 | Gene loci that amplify the expression level in the tetracycline gene expression induction system and the effect of amplification by knock-in |
JP7460643B2 (en) | 2018-10-16 | 2024-04-02 | ブルーアレル, エルエルシー | Methods for targeted insertion of DNA into genes |
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EP1403376A3 (en) * | 1993-04-21 | 2007-08-29 | The University Court Of The University Of Edinburgh | Expression of heterologous genes according to a targeted expression profile |
US5888981A (en) | 1993-06-14 | 1999-03-30 | Basf Aktiengesellschaft | Methods for regulating gene expression |
US5912411A (en) | 1993-06-14 | 1999-06-15 | University Of Heidelberg | Mice transgenic for a tetracycline-inducible transcriptional activator |
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US5464758A (en) * | 1993-06-14 | 1995-11-07 | Gossen; Manfred | Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters |
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US5695995A (en) * | 1994-05-06 | 1997-12-09 | Fred Hutchinson Cancer Research Center | Neurogenic differentiation (neurod) genes |
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US5922601A (en) | 1995-01-19 | 1999-07-13 | Biotransplant, Inc. | High efficiency gene trap selection of regulated genetic loci |
AU5557196A (en) * | 1995-04-20 | 1996-11-07 | Chiron Corporation | High efficiency ex vivo transduction of hematopoietic stem cells by recombinant retroviral preparations |
US5629159A (en) * | 1995-06-07 | 1997-05-13 | California Institute Of Technology | Immortalization and disimmortalization of cells |
US6139833A (en) | 1997-08-08 | 2000-10-31 | Lexicon Genetics Incorporated | Targeted gene discovery |
US6207371B1 (en) | 1996-10-04 | 2001-03-27 | Lexicon Genetics Incorporated | Indexed library of cells containing genomic modifications and methods of making and utilizing the same |
US5965995A (en) * | 1997-09-18 | 1999-10-12 | Allen-Bradley Company, Llc | Transient inductance tuner for motor control |
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Non-Patent Citations (3)
Title |
---|
JOURNAL OF VIROLOGY, May 1991, Vol. 65, No. 5, SORIANO et al., "Promoter Interactions in Retrovirus Vectors Introduced into Fibroblasts and Embryonic Stem Cells", pages 2314-2319. * |
See also references of EP0951533A4 * |
TRANSGENIC RESEARCH, July 1994, Vol. 3, No. 4, RIJKERS et al., "Insertional Mutagenesis in Transgenic Mice", pages 203-215. * |
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US20020115210A1 (en) | 2002-08-22 |
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US20010056581A1 (en) | 2001-12-27 |
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AU5807998A (en) | 1998-07-31 |
US20020012996A1 (en) | 2002-01-31 |
CA2276529A1 (en) | 1998-07-09 |
AU736267B2 (en) | 2001-07-26 |
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US8389795B2 (en) | 2013-03-05 |
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