Identification of germline competent chimaeras by copulatory plug genotyping.
The increasing use of ES cell lines from strains other than 129, in particular C57BL/6, has greatly reduced the time taken to generate gene knockouts on a defined genetic background. Generally, C57BL/6 ES cell lines transmit less efficiently through the germline than 129 lines; consequently the burden on animal husbandry at this stage is increased. Genotyping sperm from chimaeric males may be used to identify mice which are transmitting the manipulated allele, however it requires that the mice be culled and the sperm used for IVF. Here we describe a quick and reliable method for genotyping copulatory plugs. Males which produce a positive result can then be naturally mated. Thus far we have observed a perfect correlation between copulatory plug genotype and germline transmission, accompanied by considerable savings in mouse numbers and resources.
Wilson
Stephen
S
Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Harlow, Essex, CM19 5AD, UK.
Sheardown
Steven A
SA
D000818:Animals; D001947:Breeding; D002460:Cell Line; D002678:Chimera / Q000235:genetics*; D003307:Copulation; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells; D005260:Female; D018390:Gene Targeting / Q000379:methods; D005838:Genotype; D005854:Germ Cells; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013094:Spermatozoa