Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-07-21
2004-06-08
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S471000, C435S320100, C435S252350
Reexamination Certificate
active
06746870
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of methods for obtaining specific and stable integration of nucleic acids into chromosomes of eukaryotes. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the &PHgr;C31 integrase.
2. Background
Genetic transformation of eukaryotes often suffers from significant shortcomings. For example, it is often difficult to reproducibly obtain integration of a transgene at a particular locus of interest. Homologous recombination generally occurs only at a very low frequency. To overcome this problem, site-specific recombination systems have been employed. These methods involve the use of site-specific recombination systems that can operate in higher eucaryotic cells.
Many bacteriophage and integrative plasmids encode site-specific recombination systems that enable the stable incorporation of their genome into those of their hosts. In these systems, the minimal requirements for the recombination reaction are a recombinase enzyme, or integrase, which catalyzes the recombination event, and two recombination sites (Sadowski (1986)
J. Bacteriol
. 165: 341-347; Sadowski (1993)
FASEB J
. 7: 760-767). For phage integration systems, these are referred to as attachment (att) sites, with an attP element from phage DNA and the attB element encoded by the bacterial genome. The two attachment sites can share as little sequence identity as a few base pairs. The recombinase protein binds to both att sites and catalyzes a conservative and reciprocal exchange of DNA strands that result in integration of the circular phage or plasmid DNA into host DNA. Additional phage or host factors, such as the DNA bending protein IHF, integration host factor, may be required for an efficient reaction (Friedman (1988)
Cell
55:545-554; Finkel & Johnson (1992)
Mol. Microbiol
. 6: 3257-3265). The reverse excision reaction sometimes requires an additional phage factor, such as the x is gene product of phage &lgr; (Weisberg & Landy (1983) “Site-specific recombination in phage lambda.” In
Lambda II
, eds. Hendrix et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp.211-250; Landy (1989)
Ann. Rev. Biochem
. 58: 913-949.
The recombinases have been categorized into two groups, the &lgr; integrase (Argos et al. (1986)
EMBO J
. 5: 433-44; Voziyanov et al. (1999)
Nucl. Acids Res
. 27: 930-941) and the resolvase/invertase (Hatfull & Grindley (1988) “Resolvases and DNA-invertases: a family of enzymes active in site-specific recombination” In
Genetic Recombination
, eds. Kucherlipati, R., & Smith, G. R. (Am. Soc. Microbiol., Washington D.C.), pp. 357-396) families. These vary in the structure of the integrase enzymes and the molecular details of their mode of catalysis (Stark et al. (1992)
Trends Genetics
8: 432-439). The temperate Streptomyces phage &PHgr;C31 encodes a 68 kD recombinase of the latter class. The efficacy of the &PHgr;C31 integrase enzyme in recombining its cognate attachment sites was recently demonstrated in vitro and in vivo in recA mutant
Escherichia coli
(Thorpe & Smith (1998)
Proc. Nat'l. Acad. Sci. USA
95: 5505-5510). The &PHgr;C31 integration reaction is simple in that it does not require a host factor and appears irreversible, most likely because an additional phage protein is required for excision. The phage and bacterial att sites share only three base pairs of homology at the point of cross-over. This homology is flanked by inverted repeats, presumably binding sites for the integrase protein. The minimal known functional size for both attB and attP is ~50 bp.
The Cre-lox system of bacteriophage P1, and the FLP-FRT system of
Saccharomyces cerevisiae
have been widely used for transgene and chromosome engineering in animals and plants (reviewed by Sauer (1994)
Curr. Opin. Biotechnol
. 5: 521-527; Ow (1996)
Curr. Opin. Biotechnol
. 7: 181-186). Other systems that operate in animal or plant cells include the following: 1) the R-RS system from
Zygosaccharomyces rouxii
(Onouchi et al. (1995)
Mol. Gen. Genet
. 247: 653-660), 2) the Gin-gix system from bacteriophage Mu (Maeser & Kahmann (1991)
Mol. Gen. Genet
. 230: 170-176) and, 3) the &bgr; recombinase-six system from bacterial plasmid pSM19035 (Diaz et al. (1999)
J. Biol. Chem
. 274: 6634-6640). By using the site-specific recombinases, one can obtain a greater frequency of integration.
However, these five systems suffer from a significant shortcoming. Each of these systems have in common the property that a single polypeptide recombinase catalyzes the recombination between two sites of identical or nearly identical sequences. The product-sites generated by recombination are themselves substrates for subsequent recombination. Consequently, recombination reactions are readily reversible. Since the kinetics of intramolecular interactions are favored over intermolecular interactions, these recombination systems are efficient for deleting rather than integrating DNA. Thus, a need exists for methods and systems for obtaining stable site-specific integration of transgenes. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for obtaining stable, site-specific recombination in a eukaryotic cell. Unlike previously known methods for site-specific recombination, the recombinants obtained using the methods of the invention are stable. The recombination reaction is not reversible.
The methods involve providing a eukaryotic cell that comprises a first recombination site and a second recombination site, which second recombination site can serve as a substrate for recombination with the first recombination site. The first and the second recombination sites are contacted with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, thereby forming one or two hybrid recombination sites. Significantly, the recombinase polypeptide is one that can mediate site-specific recombination between the first and second recombination sites, but cannot mediate recombination between the two hybrid recombination sites in the absence of an additional phage-produced factor that is not present in the eukaryotic cell. Either or both of the recombination sites can be present in a chromosome of the eukaryotic cell. In some embodiments, one of the recombination sites is present in the chromosome and the other is included within a nucleic acid that is to be integrated into the chromosome.
The invention also provides eukaryotic cells that contain a prokaryotic recombinase polypeptide or a nucleic acid that encodes a prokaryotic recombinase. In these embodiments, the recombinase is one that can mediate site-specific recombination between a first recombination site and a second recombination site that can serve as a substrate for recombination with the first recombination site, but in the absence of an additional factor that is not present in the eukaryotic cell cannot mediate recombination between two hybrid recombination sites that are formed upon recombination between the first recombination site and the second recombination site. In presently preferred embodiments, the cells of the invention include a nucleic acid that has a coding sequence for a recombinase polypeptide. The recombinase coding sequence is preferably operably linked to a promoter that mediates expression of the recombinase-encoding polynucleotide in the eukaryotic cell. The eukaryotic cells of the invention can be an animal cell, a plant cell, a yeast cell or a fungal cell, for example.
In additional embodiments, the invention provides methods for obtaining a eukaryotic cell having a stably integrated transgene. These methods involve introducing a nucleic acid into a eukaryotic cell that comprises a first recombination site, wherein the nucleic acid comprises the transgene of interest and a second recombination site which can serve as a substrate for recombination with the first recombination site. The first and second recombinati
Calendar Richard
Ow David W.
Thomason Lynn
Katcheves Konstantina
The Regents of the University of California
Townsend and Townsend / and Crew LLP
Yucel Remy
LandOfFree
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