To create heterozygous, conditional homozygous, homozygous knockout and rescue Bptf ES cell lines we transiently expressed Cre followed by retargeting. The piCre expression vector was electroporated into clone 7010 and conversion from Bptf/BptfFloxedNeo to Bptf/BptfΔexon2 was first screened for by PCR then confirmed by Southern blotting. Three clones were identified using this strategy, and clone 7004 was used for subsequent targeting. The wild type allele in the Bptf/BptfΔexon2 from clone 7004 was then retargeted using the Bptf exon 2 targeting vector. Individual conditional homozygous clones were first screened for by PCR then confirmed by Southern blotting. Two BptfFloxedNeo/BptfΔexon2 clones were obtained and named H12 and B19. The piCre expression vector was electroporated into H12 and B19 and conversion from BptfFloxedNeo/BptfΔexon2 to BptfΔexon2/BptfΔexon2 was first screened for by PCR then confirmed by Southern blotting. A total of 9 and 5 homozygous knockout clones were obtained from the H12 and B19 parental lines respectively. The karyotype of the knockout lines were confirmed using Giemsa staining and lines with an anuploid karyotype were discarded. ES cells were maintained on mitomycin C treated primary
