Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-07-19
2004-11-23
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S029000, C435S235100, C435S252300, C435S320100, C435S091410, C435S091420, C435S325000, C435S455000, C435S468000, C435S471000, C536S023100
Reexamination Certificate
active
06821759
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to methods of modifying genes with specificity in recombination deficient cells by transiently enabling homologous recombination in the cells. Included in the invention are conditional replication shuttle vectors which bestow transient recombination capabilities to an otherwise recombination deficient cell. The independent origin based cloning vectors containing the modified genes and methods of using the independent origin based cloning vectors containing the modified genes are also included in the present invention. In particular, high throughput methodology is provided for generating the modified the independent origin based cloning vectors.
BACKGROUND OF THE INVENTION
Functional analyses of genes in vivo frequently involve the introduction of modified genomic DNA into the germline to generate transgenic animals [Jaenisch et al.,
Science
240:1468 (1985); Brinster,
Cell
41:343 (1985)]. The genomic DNA sequences containing introns and essential regulatory sequences have been shown to be expressed in vivo in cases where simple cDNA constructs cannot be expressed [Brinster et al.,
Proc.Natl.Acad.Sci.
85:836-840 (1988)]. Furthermore, the size of the genomic DNA that can be readily manipulated in vitro and introduced into the germline can be a critical determinant of the outcome of the functional analysis of a gene since elements that are important for high level, tissue specific and position-independent expression of the transgene may be located at a long distance from the gene itself [Dillon et al.,
Trends Genet.
9:134 (1993); Kennison,
Trends Genet.
9:75 (1993); Wilson et al.,
Annu.Rev. Cell.Biol.
6:679 (1990)].
On the other hand, the use of such large genomic transgenes has several practical problems. For example, the size of the transgene is presently limited due to constraints on the sequence length that can be cloned and stably maintained in a conventional plasmid or a cosmid. Thus DNA sequences suspected of being nonessential are often omitted when designing the constructs to be transferred because of the size limitation. In addition, in vitro manipulations of large DNAs oftentimes lead to mechanical shear [Peterson et al.,
TIG
13:61-66].
Yeast artificial chromosomes (YACs) allow large genomic DNA to be modified and used for generating transgenic animals [Burke et al.,
Science
236:806; Peterson et al.,
Trends Genet.
13:61 (1997); Choi, et al.,
Nat. Genet.,
4:117-223 (1993), Davies, et al.,
Biotechnology
11:911-914 (1993), Matsuura, et al.,
Hum. Mol. Genet.,
5:451-459 (1996), Peterson et al.,
Proc. Natl. Acad. Sci.,
93:6605-6609 (1996); and Schedl, et al.,
Cell,
86:71-82 (1996)]. Other vectors also have been developed for the cloning of large segments of mammalian DNA, including cosmids, and bacteriophage P1 [Sternberg et al.,
Proc. Natl. Acad. Sci. U.S.A.,
87:103-107 (1990)]. YACs have certain advantages over these alternative large capacity cloning vectors [Burke et al.,
Science,
236:806-812 (1987)]. The maximum insert size is 35-30 kb for cosmids, and 100 kb for bacteriophage P1, both of which are much smaller than the maximal insert for a YAC. However, there are several critical limitations in the YAC system including difficulties in manipulating YAC DNA, chimerism and clonal instability [Green et al.,
Genomics,
11:658 (1991); Kouprina et al.,
Genomics
21:7 (1994); Larionov et al.,
Nature Genet.
6:84 (1994)]. As a result, generating transgenic mice with an intact YAC remains a challenging task [Burke et al.,
Science
236:806; Peterson et al.,
Trends Genet.
13:61 (1997)].
An alternative to YACs are
E. coli
based cloning systems based on the
E. coli
fertility factor that have been developed to construct large genomic DNA insert libraries. They are bacterial artificial chromosomes (BACs) and P-1 derived artificial chromosomes (PACs) [Mejia et al.,
Genome Res.
7:179-186 (1997); Shizuya et al.,
Proc. Natl. Acad. Sci.
89:8794-8797 (1992);Ioannou et al.,
Nat. Genet.,
6:84-89 (1994); Hosoda et al.,
Nucleic Acids Res.
18:3863 (1990)]. BACs are based on the
E. coli
fertility plasmid (F factor); and PACs are based on the bacteriophage P1. The size of DNA fragments from eukaryotic genomes that can be stably cloned in
Escherichia coli
as plasmid molecules has been expanded by the advent of PACs and BACs. These vectors propagate at a very low copy number (1-2 per cell) enabling genomic inserts up to 300 kb in size to be stably maintained in recombination deficient hosts (most clones in human genomic libraries fall within the 100-200 kb size range). The host cell is required to be recombination deficient to ensure that non-specific and potentially deleterious recombination events are kept to a very minimum. As a result, libraries of PACs and BACs are relatively free of the high proportion of chimeric or rearranged clones typical in YAC libraries, [Monaco et al.,
Trends Biotechnol
12:280-286 (1994); Boyseu et al.,
Genome Research,
7:330-338 (1997)]. In addition, isolating and sequencing DNA from PACs or BACs involves simpler procedures than for YACs, and PACs and BACs have a higher cloning efficiency than YACs [Shizuya et al.,
Proc. Natl. Acad. Sci.
89:8794-8797 (1992);Ioannou et al.,
Nat. Genet.,
6:84-89 (1994); Hosoda et al.,
Nucleic Acids Res.
18:3863 (1990)]. Such advantages have made BACs and PACs important tools for physical mapping in many genomes [Woo et al.,
Nucleic Acids Res.,
22:4922 (1994); Kim et al.,
Proc.Natl.Acad.Sci.
93:6297-6301 (1996); Wang et al.,
Genomics
24:527 (1994); Wooster et al.,
Nature
378:789 (1995)]. Furthermore, the PACs and BACs are circular DNA molecules that are readily isolated from the host genomic background by classical alkaline lysis [Bimboim et al.,
Nucleic Acids Res.
7:1513-1523 (1979].
Functional characterization of a gene of interest contained by a PAC or BAC clone generally entails transferring the DNA into a eukaryotic cell for transient or long-term expression. A transfection reporter gene, e.g. a gene encoding lacZ, together with a selectable marker, e.g., neo, can be inserted into a BAC [Mejia et al.,
Genome Res.
7:179-186 (1997)]. Transfected cells can be then detected by staining for X-Gal to verify DNA uptake. Stably transformed cells are selected for by the antibiotic G418.
However, while PACs and BACs have cloning capacities up to 350 kb, performing homologous recombination to introduce mutations into a gene of interest has not been demonstrated [Peterson et al.,
TIG
13:61-66]. Indeed, although BACs or PACs have become an important source of large genomic DNA in genome research, there are still no methods available to modify the BACs or PACs. Furthermore, no germline transmission of intact BACs or PACs in transgenic mice have been reported. These, as well as other disadvantages of BACs and PACs greatly limit their potential use for functional studies. Therefore, there is a need for an improved cloning vector for germline transmission of selected genes in transgenic animals. More particularly there is a need for a cloning vector that has the capacity to contain greater than 100 kilobases of DNA, which can be readily manipulated and isolated, but still can be stably stored in libraries relatively free of rearranged clones. In addition, there is a need to provide methodology for generating such cloning vectors. There is also a need to apply such vectors to improve the results of the methods of gene transfer used in gene targeting, for creating animal models for diseases due to a dominant mutated allele, e.g., Huntington's disease, and for overexpressing in vivo proteins encoded by genes having an unknown function in order to determine the biological role of such genes.
Gene targeting has been used in various systems, from yeast to mice, to make site specific mutations in the genome. Gene targeting is not only useful for studying function
Gong Shiaoching
Heintz Nathaniel
Model Peter
Yang Xiangdong W.
Leffers Gerry
The Rockefeller University
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