Construction of endogenous affinity-tagged Oct4/Pou5f1 alleles by gene-targeted homologous recombination in embryonic stem cells. (A) Each of four different gene-targeting constructs comprising dual-affinity tags was constructed to target the endogenous Oct4/Pou5f1 allele. NTAP (protein A calmodulin-binding peptide), NSC (S peptide calmodulin-binding peptide), NBH (biotin acceptor peptide-HIS) or NFH (FLAG-HIS) was inserted at the ATG translation start site of Oct4. The wild-type Oct4 allele gives an 11.7-kb restriction fragment during digestion with EcoRI, whereas correctly targeted homologous recombination results in a mutant Oct4 allele that gives a 6.5-kb fragment instead. These fragments were detected using an external probe that lies between the EcoRI site and the HpaI site. (B) Southern blot analysis of embryonic stem (ES) cell colonies after endogenous Oct4 modification with the targeting construct for introduction of NTAP tag. Wild-type (WT) Oct4 BAC and NTAP-Oct4 BAC were used as positive controls for the wild-type allele fragment and the mutant allele fragment, respectively. The probe used is indicated in Figure 1A. (C) Diagram showing the proteins that are expressed by the respective modified Oct4 alleles. NBH, N-terminal biotin acceptor peptide HIS; NFH, N-terminal FLAG HIS; NSC, N-terminal S peptide calmodulin-binding peptide; NTAP, N-terminal tandem affinity purification; BAP, biotin acceptor peptide; CBP, calmodulin-binding peptide; S, S peptide; TEV, tobacco etch virus.
Cheong et al. Stem Cell Research & Therapy 2011 2:26 doi:10.1186/scrt67