Recombineering-based procedure for creating Cre/loxP conditional knockouts in the mouse.
Gene targeting in the mouse is an essential tool for studying gene function and creating models of human disease. The method described in this unit takes advantage of bacterial artificial chromosomes, Cre/loxP and FLPe/FRT systems, and recently evolved recombineering approaches to simplify the preparation of targeting constructs for generation of conditional knockout (CKO) animals. This method has been used to generate >30 CKO constructs, most of them successfully used to target mouse ES cells and establish mutant mice. Design and preparation of the CKO construct, as well as step-wise troubleshooting guidelines, are described in detail.
Bouvier
Jason
J
UNC-Chapel Hill, Chapel Hill, North Carolina, USA.
Cheng
Jr-Gang
JG
D000818:Animals; D001287:Attachment Sites, Microbiological; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D053595:Embryonic Stem Cells / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000235:genetics* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic
D004274:DNA, Recombinant; C045073:Cre recombinase; D019426:Integrases