Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-12-11
2003-02-25
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S462000, C435S455000, C435S320100, C435S325000, C536S023100, C536S023500, C514S04400A, C424S093200
Reexamination Certificate
active
06524856
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods for targeting sequence modifications in one or more genes of a related family of genes using enhanced homologous recombination techniques. The invention also relates to compositions and methods for isolating and identifying novel members of homologous sequences families. These techniques may be used to create animal or plant models of disease as well as to identify new targets for drug or pathogen screening.
BACKGROUND
Homologous recombination (or general recombination) is defined as the exchange of homologous segments anywhere along a length of two DNA molecules. An essential feature of general recombination is that the enzymes responsible for the recombination event can presumably use any pair of homologous sequences as substrates, although some types of sequence may be favored over others. Both genetic and cytological studies have indicated that such a crossing-over process occurs between pairs of homologous chromosomes during meiosis in higher organisms.
Alternatively, in site-specific recombination, exchange occurs at a specific site, as in the integration of phage &lgr; into the
E. coli
chromosome and the excision of &lgr; DNA from it. Site-specific recombination involves specific inverted repeat sequences; e.g. the Cre-loxP and FLP-FRT systems. Within these sequences there is only a short stretch of homology necessary for the recombination event, but not sufficient for it. The enzymes involved in this event generally cannot recombine other pairs of homologous (or nonhomologous) sequences, but act specifically.
Although both site-specific recombination and homologous recombination are useful mechanisms for genetic engineering of DNA sequences, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence. Site-specific recombination has been proposed as one method to integrate transfected DNA at chromosomal locations having specific recognition sites (O'Gorman et al. (1991)
Science
251: 1351; Onouchi et al. (1991)
Nucleic Acids Res
. 19: 6373). Unfortunately, since this approach requires the presence of specific target sequences and recombinases, its utility for targeting recombination events at any particular chromosomal location is severely limited in comparison to targeted general recombination.
Homologous recombination has also been used to create transgenic plants and animals. Transgenic organisms contain stably integrated copies of genes or gene constructs derived from another species in the chromosome of the transgenic organism. In addition, gene targeted animals can be generated by introducing cloned DNA constructs of the foreign genes into totipotent cells by a variety of methods, including homologous recombination. For example, animals that develop from genetically altered totipotent cells can contain the foreign gene in all somatic cells and also in germ-line cells. Currently methods for producing transgenic and targeted animals have been performed on totipotent embryonic stem cells (ES) and with fertilized zygotes. ES cells have an advantage in that large numbers of cells can be manipulated easily by homologous recombination in vitro before they are used to generate targeted animals. Currently, however, only embryonic stem cells from mice have been shown to contribute to the germ line. Alternatively, DNA can also be introduced into fertilized oocytes by micro-injection into pronuclei which are then transferred into the uterus of a pseudo-pregnant recipient animal to develop to term. The ability of mammalian and human cells to incorporate exogenous genetic material into genes residing on chromosomes has demonstrated that these cells have the general enzymatic machinery for carrying out homologous recombination required between resident and introduced sequences. These targeted recombination events can be used to correct mutations at known sites, replace genes or gene segments with defective ones, or introduce foreign genes into cells.
Traditionally, exogenous sequences transferred into eukaryotic cells undergo homologous recombination with homologous endogenous sequences only at very low frequencies, and are so inefficiently recombined that large numbers of cells must be transfected, selected, and screened in order to generate a desired correctly targeted homologous recombinant (Kucherlapati et al. (1984)
Proc. Natl. Acad. Sci. (U.S.A.)
81: 3153; Smithies, 0. (1985)
Nature
317: 230; Song et al. (1987)
Proc. Natl. Acad. Sci. (U.S.A.)
84: 6820; Doetschman et al. (1987)
Nature
330: 576; Kim and Smithies (1988)
Nucleic Acids Res
. 16: 8887; Doetschman et al. (1988)
Proc. Natl. Acad. Sci. (USA)
85: 8583; Koller and Smithies (1989)
Proc. Natl. Acad. Sci. (USA)
86: 8932; Shesely et al. (1991)
Proc. Natl. Acad. Sci. (U.S.A.)
88: 4294; Kim et al. (1991)
Gene
103: 227, which are incorporated herein by reference).
Several proteins or purified extracts having the property of promoting homologous recombination (i.e., recombinase activity) have been identified in prokaryotes and eukaryotes (Cox and Lehman (1987)
Ann. Rev. Biochem
. 56: 229; Radding, C. M. (1982)
ANNU. Rev. Genet
. 16:405; Madiraju et al. (1988)
Proc. Natl. Acad. Sci. (U.S.A.)
85: 6592; McCarthy et al. (1988)
Proc. Natl. Acad. Sci. (U.S.A.)
85: 5854; Lopez et al. (1987)
Nucleic Acids Res
. 15:5643,6813, which are incorporated herein by reference). These general recombinases presumably promote one or more steps in the formation of homologously-paired intermediates, strand-exchange, gene conversion, and/or other steps in the process of homologous recombination.
The frequency of homologous recombination in prokaryotes is significantly enhanced by the presence of recombinase activities. Several purified proteins catalyze homologous pairing and/or strand exchange in vitro, including:
E. coli
recA protein, the T4 uvsX protein, the rec1 protein from
Ustilago maydis
, and Rad51protein from
S. cerevisiae
(Sung et al., Science 265:1241 (1994)) and human cells (Baumann et al., Cell 87:757 (1996)). Additional members of this protein family have been identified by homology and function including Rad51A, B, C, D & E. Dosanjh, et cl., (1998) Nucleic Acid Res. 26:1179-1184 and dmc1. Recombinases and dmel, like the recA protein of
E. coli
are proteins which promote strand pairing and exchange. The most studied recombinase to date has been the recA recombinase of
E. coli
, which is involved in homology search and strand exchange reactions (see, Cox and Lehman (1987)
ANNU. Rev. Biochem
. 56:229. RecA is required for induction of the SOS repair response, DNA repair, and efficient genetic recombination in
E coli
. RecA can catalyze homologous pairing of a linear duplex DNA and a homologous single strand DNA in vitro. In contrast to site-specific recombinases, proteins like recA which are involved in general recombination recognize and promote pairing of DNA structures on the basis of shared homology, as has been shown by several in vitro experiments (Hsieh and Camerini-Otero (1989)
J. Biol. Chem
. 264: 5089; Howard-Flanders et al. (1984)
Nature
309: 215; Stasiak et al. (1984)
Cold Spring Harbor Symp. Quant. Biol
. 49: 561; Register et al. (1987)
J. Biol. Chem
. 262: 12812). Several investigators have used recA protein in vitro to promote homologously paired triplex DNA (Cheng et al. (1988)
J. Biol. Chem
. 263: 15110; Ferrin and Camerini-Otero (1991)
Science
354: 1494; Ramdas et al. (1989)
J. Biol Chem
. 264: 11395; Strobel et al. (1991)
Science
254: 1639; Hsieh et al. (1990)
Genes Dev
. 4:1951; Rigas et al. (1986)
Proc. Natl. Acad. Sci. (U.S.A.)
83: 9591; and Camerini-Otero et al. U.S. Pat. No. 7,611,268, which are incorporated herein by reference).
Recent advances have resulted in techniques allowing enhanced homologous recombination (EHR) using recombinases such as recA and Rad51 and single-stranded nucleic acids that have sequence heterolog
Pati Sushma
Zarling David A.
Zeng Hong
Capps Nancy B.
Kaushal Sumesh
Pangene Corporation
Priebe Scott D.
Trecartin Richard R.
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