Cre-mediated recombination at the murine whey acidic protein (mWAP) locus.
The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal "bioreactor" models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland.
Rucker
E B
EB
Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station 77843, USA.
Piedrahita
J A
JA
D000818:Animals; D019149:Bioreactors; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D018274:Electroporation; D019426:Integrases / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008894:Milk Proteins / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D013234:Stem Cells / Q000166:cytology*; D019076:Transgenes / Q000235:genetics*; D014764:Viral Proteins
D008894:Milk Proteins; D014764:Viral Proteins; C024471:whey acidic proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases