title: Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems.
abstract: Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).
authors.affiliation: Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Minorikawa
authors.forename: Manabu
authors.initials: M
authors.lastname: Nakayama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D000071317:Nanog Homeobox Protein; D016133:Polymerase Chain Reaction; D045522:Recombinases / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D045522:Recombinases