Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos.
Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. itomasanori@med.toho-u.ac.jp
Masanori
M
Ito
Keitaro
K
Yamanouchi
Kunihiko
K
Naito
Michele P
MP
Calos
Hideaki
H
Tojo
D000818:Animals; D030801:Animals, Genetically Modified; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D018390:Gene Targeting; D019426:Integrases / Q000378:metabolism*; D007763:Lac Operon; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D001616:beta-Galactosidase / Q000378:metabolism
C045073:Cre recombinase; D019426:Integrases; D001616:beta-Galactosidase