Mutational analysis of loxP sites for efficient Cre-mediated insertion into genomic DNA.
The Cre/loxP system has been used in transgenic models primarily to excise DNA flanked by loxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant loxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing lambda integrase, mutant loxP sites were placed at the E. coli attB site and the excision-insertion ratios of incoming DNA plasmids carrying a second, complementary mutant loxP site were determined. Comparison of 50 mutant loxP sites combinations to the native loxP site revealed that mutations to the inner 6 bp of the Cre binding domain severely inhibited recombination, while those in the outer 8 bps were more tolerated. The most efficient loxP combinations resulted in 1421-fold and 1529-fold increases in relative integration rates over wild-type loxP sites. These loxP mutants could be exploited for site-directed "tag and insert" recombination experiments.
Thomson
James G
JG
Department of Veterinary Anatomy and Public Health, Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas, USA.
Rucker
Edmund B
EB
Piedrahita
Jorge A
JA
D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers; D018274:Electroporation; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D017931:DNA Primers; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases