Vectors and methods for the mutagenesis of mammalian genes

Details Vectors and methods for the mutagenesis of mammalian genes Vectors and methods for the mutagenesis of mammalian genes

1997-12-31

2001-05-08

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C435S440000, C435S455000

Reexamination Certificate

active

06228639

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to retroviral vectors and their use in methods of mammalian gene mutagenesis.
Eukaryotic genomes are estimated to contain 6,000-80,000 genes (Collins, Proc. Natl. Acad. Sci. USA 92:10821-10823 (1995)). Even in the best characterized organisms, the function of the majority of these genes is unknown. In addition, relatively little information is available concerning the fraction of the genome that is expressed in particular cell types or the cellular processes in which specific gene products participate. In an attempt to decipher genes' functions, large scale mutagenesis screens have been developed and have proven instrumental in unraveling the roles of certain genes in organisms such as
Drosophila melanogaster
(Nusslein-Volhard and Wieschaus, Nature 287:795-801 (1980); Ballinger and Benzer, Proc. Natl. Acad. Sci. USA 86:9402-9406 (1989); Kaiser and Goodwin, Proc. Natl. Acad. Sci. USA 87:1686-1690 (1990); and Spradling et al., Proc. Natl. Acad. Sci. USA 92:10824-10830 (1995)),
Caenorhabditis elegans
(Hirsh and Vanderslice, Dev Biol. 49:220-235 (1976); and Zwaal et al., Proc. Natl. Acad. Sci. USA 90:7431-7435 (1993)), Zebrafish (Solnica-Krezel et al., Genetics 136:1401-1420 (1994); and Riley and Grunwald, Proc. Natl. Acad. Sci. USA 92:5997-6001 (1995)), Arabidopsis (Jurgens et al., Development Suppl. 1:27-38 (1991); Mayer et al., Nature 353:402-407 (1991); and Sundaresan et al., Genes Dev. 9:1797-1810 (1995)), Maize (Scanlon et al., Genetics 136:281-294 (1994); and Osborne and Baker, Curr. Opin. Cell Biol. 7:406-413 (1995)), and
Saccharomyces cerevisiae
(Burns et al., Genes Dev. 8: 1087-1105 (1994); and Chun and Goebl, Genetics 142:30-50 (1996)). In mammals, however, these approaches have generally been limited by the large genome size and the development of the embryo inside a mother's uterus.
Some progress has been made in understanding mammalian gene function as a result of the development of mouse embryonic stem (ES) cell technology. This technology has significantly altered the field of mammalian genetics by allowing the bulk of genetic manipulations to be executed in vitro (Evans and Kaufman, Nature 292:154-156 (1981); Bradley et al., Nature 309:255-256 (1984); and Robertson, Trends Genet. 2:9-13 (1986)). This is possible because mouse ES cells are pluripotent, that is, they have the ability to generate entirely ES cell-derived animals. Accordingly, gene inactivation in mouse ES cells and subsequent generation of “knock-out” (KO) mice is a powerful method for gaining information about the function of a gene in a whole animal system. If desired, genetic alterations, such as gene KOs which inactivate genes, may be introduced into these cells, and their consequences may be studied in the whole animal (Jaenisch, Science 240:1468-1474 (1988); and Rossant and Nagy, Nat. Med. 1:592-594 (1995)).
Currently, the available mouse mutagenesis methodologies are somewhat limited in their general utility as gene function screening systems. Gene targeting, the most widely used approach, is laborious and time consuming (Capecchi, Science 244:1288-1292 (1989)). And gene trap and chemical/radiation induced mutagenesis are generally restricted in their targets (Gossler et al., Science 244:463-465 (1989); Friedrich and Soriano, Genes Dev. 5:1513-1523 (1991); Skarnes et al., Genes Dev. 6:903-918 (1992); von Melchner et al., Genes Dev. 6:919-927 (1992); Reddy et al., Proc. Natl. Acad. Sci. USA 89:6721-6725 (1992); Takeuchi et al., Genes Dev. 9:1211-1222 (1995); and Takahashi et al., Science 264:1724-1733 (1994)). 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); Skames 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.
SUMMARY OF THE INVENTION
In general, the invention features a method for mutagenizing a mammalian gene, the method involving introducing into a mammalian cell (for example, a stem cell, such as an embryonic stem cell) a retroviral vector, the vector including a splice acceptor sequence, a transcription termination sequence, and retroviral packaging and integration sequences, the introducing step being carried out under conditions which allow the vector to integrate into the genome of the cell.
In preferred embodiments, the retroviral vector includes packaging and integration sequences derived from a Moloney murine leukemia virus sequence; the retroviral vector further includes a reporter gene whose expression is under the control of a mammalian cell promoter, the promoter being operably linked to the reporter gene upon integration of the vector into the genome of the mammalian cell; the reporter gene encodes a regulatory protein, the regulatory protein being capable of modulating the expression of a detectable gene; the regulatory protein is a tetracycline repressor fused to an activator protein (for example, VP16); the retroviral vector further includes a DNA sequence encoding a constitutively expressed marker gene, the marker gene being detectable in a mammalian cell; the marker gene is a green fluorescent protein (for example, a green fluorescent having increased cellular fluorescence relative to a wild type green fluorescent protein); the green fluorescent protein is fused to a mammalian selectable marker; the mammalian selectable marker encodes neomycin resistance; the retroviral vector further includes a recognition sequence derived from a yeast VDE DNA endonuclease; the retroviral vector further includes a sequence which is recognized by a recombinase enzyme (for example, a loxP sequence); the mammal is a mouse; and the cell is an embryonic stem cell.
In a related embodiment, the invention features a retroviral vector which includes a splice acceptor sequence, a transcription termination sequence, and retroviral packaging and integration sequences. In preferred embodiments, the retroviral vector includes packaging and integration sequences derived from a Moloney murine leukemia virus sequence; the retroviral vector further includes a reporter gene whose expression is under the control of a mammalian cell promoter, the promoter being operably linked to the reporter gene upon integration of the vector into the genome of the mammalian cell; the reporter gene encodes a regulatory protein, the regulatory protein being capable of modulating the expression of a detectable gene; the regulatory protein is a tetracycline repressor fused to an activator protein (for example, VP16); the detectable gene includes an operably linked tetracycline operator; the retroviral vector further includes a DNA sequence encoding a constitutively expressed marker gene, the marker gene being detectable in a mammalian cell; the marker gene is a green fluorescent protein (for example, a green fluorescent protein having increased cellular fluorescence relative to a wild type green fluorescent protein); the green fluorescent protein is fused to a mammalian selectable marker; the mammalian selectable marker encodes neomycin resistance; the retroviral vector further includes a recognition sequence derived from a yeast VDE DNA endonuclease; and the retroviral vector further includes a sequence which is recognized by a recombinase enzyme (for example, a loxP sequence).
In other related embodiments, 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 b

Affiliated with

Also associated with

Rating

Vectors and methods for the mutagenesis of mammalian genes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Vectors and methods for the mutagenesis of mammalian genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Vectors and methods for the mutagenesis of mammalian genes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2509652