Fig 1. Generation of ptip^−/− ES cells.

PCR genotyping of ES cells with allele specific primer pairs. A) Schematic of the different alleles used in this study. The floxed allele contains loxP sites flanking exon 1 (ex1); a single flip recombinase site is also present (open triangle). The germline null allele deletes exons 1–3 and still contains PGK-neo, as described previously. Primers P1 and P2 were used to detect the wild-type and floxed alleles, primers P1 and P3 detect the Cre excised allele, and primers n1 and n2 detect the neo-containing null allele. B) Wild-type and ES cell lines derived from a ptip^fl/fl x ptip^fl/− mating were genotyped with allele specific primer pairs before Cre mediated excision. Top panel indicates the wild-type (+) and floxed alleles. The bottom panel shows the germline null allele. C) Genotype of ES cells after infection with Cre expressing adenovirus (Ad-Cre). As indicated, cells were cultured as embryoid bodies, without feeders, or as ES cells on feeders and LIF. Note, wild-type allele is due to feeder cells. D) Relative mRNA expression determined by real time qPCR. Parental ptip^fl/− ES cells (Pa), ptip^−/− derivatives (Ad-Cre1, Ad-Cre2), and parental cells infected with Ad only are shown. E) PTIP protein expression as determined by Western blotting from ptip^fl/− ES cells infected with the control (Ad) or Cre (Ad-Cre) expressing adenovirus as indicated.