ESC lines were thawed into M15+LIF medium and cultured for 2 d prior to targeting. CHC targeting was performed as described previously (Dow et al. 2012 (link)). The Pdx1 ESC line was cultured in KOSR+2i medium (Gertsenstein et al. 2010 (link)) starting immediately after targeting and also throughout selection with hygromycin (Roche). The p48 ESC line was kept in M15+LIF and changed to KOSR+2i no earlier than 48 h prior to blastocyst injection.
Correct integration of the targeting vector into the CHC locus was confirmed by PCR as described previously (Dow et al. 2012 (link)). To rule out additional random integrations of the targeting vector in the genome, we performed a TaqMan copy number assay for GFP (Invitrogen) according to the manufacturer's instructions on a ViiA7 RT–PCR machine (Life Technologies). Functionally, ESC clones were tested by treatment with and without adenoviral Cre recombinase (University of Iowa) to activate the latent rtTA3 within the CAGs-LSL-RIK allele in the adenoviral Cre-treated ESCs and by subsequent addition of dox to the growth medium for 3 d, and GFP induction was detected by flow cytometry to determine activation of the targeted CHC locus.
Correct integration of the targeting vector into the CHC locus was confirmed by PCR as described previously (Dow et al. 2012 (link)). To rule out additional random integrations of the targeting vector in the genome, we performed a TaqMan copy number assay for GFP (Invitrogen) according to the manufacturer's instructions on a ViiA7 RT–PCR machine (Life Technologies). Functionally, ESC clones were tested by treatment with and without adenoviral Cre recombinase (University of Iowa) to activate the latent rtTA3 within the CAGs-LSL-RIK allele in the adenoviral Cre-treated ESCs and by subsequent addition of dox to the growth medium for 3 d, and GFP induction was detected by flow cytometry to determine activation of the targeted CHC locus.
