Dna Recombination In Eukaryotic Cells By The Bacteriophage Phic31 Recombination System

Tech ID: 18679 / UC Case 1999-099-0

Abstract

This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The method is an alternative to the widely used CRE-LOX method.

The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the phiC31 recombinase, that can mediate recombination between the recombination sites, but not between hybrid recombination sites that are formed upon the recombination. Thus, the recombination is irreversible in the absence of additional factors. Eukaryotic cells that contain the recombinase polypeptides, or genes that encode the recombinases, are also provided.

See also:

MGG Molecular Genetics and Genomics. August, 2001. 265(6):1031-1038.

Thomason, L. C.; Calendar, R.; Ow, D. W.

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophagevariant phiC31 site-specific recombination system.

Abstract:

The site-specific recombination system used by the Streptomyces bacteriophage variant phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the variant phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBXattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The variant phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the variant phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the variant phiC31 recombination provides new opportunities for directing transgene Y and chromosome rearrangements

in eukaryotic systems.

Applications

Alternative to the CRE-LOX system

Advantages

Alternative to the CRE-LOX system for knock-out mutation generation

Patent Status

Country Type Number Dated Case
United States Of America Issued Patent 8,129,598 02/21/2012 1999-099
United States Of America Issued Patent 6,746,870 06/08/2004 1999-099
 

Contact

Learn About UC TechAlerts - Save Searches and receive new technology matches

Other Information

Keywords

target validation

Categorized As