title: Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies.
abstract: Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.
authors.affiliation: Retinal Circuits Development and Genetics Unit, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Katherine
authors.initials: K
authors.lastname: Chuang
authors.affiliation: Retinal Circuits Development and Genetics Unit, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Eileen
authors.initials: E
authors.lastname: Nguyen
authors.affiliation: Retinal Circuits Development and Genetics Unit, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Sergeev
authors.affiliation: Retinal Circuits Development and Genetics Unit, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 badeatc@mail.nih.gov.
authors.forename: Tudor C
authors.initials: TC
authors.lastname: Badea
mesh_terms: D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015723:Gene Library; D023061:Gene Order; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D019426:Integrases / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D045522:Recombinases / Q000378:metabolism
chemical_list: C546029:Dre recombinase, E coli; D029968:Escherichia coli Proteins; D045522:Recombinases; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase
keywords: Cre recombination; Dre recombinase; gene targeting; site specific recombination