FIGURE 1.

Generation of TK-hESCs. A, the endogenous NANOG locus in hESCs. Open boxes indicate the 5′ and 3′ UTRs of the human NANOG gene. Filled boxes indicate the NANOG open reading frame. The stop codon (TGA) of the NANOG gene and the location of the probe for Southern blotting are indicated. The size of the WT BamHI restriction fragment is shown. B, BAC-based targeting vector. The IRES-TKSR39-IRES-Puro-IRES-EGFP expression cassette was inserted into the BAC vector ∼100 bp downstream of NANOG TGA stop codon by recombineering. The sizes of homologous arms are indicated. C, the configuration of the knock-in allele. The size of mutant BamHI restriction fragment is indicated. D, Southern blotting analysis of the knock-in TK-hESC (3IA) clone in H9 hESC line. Genomic DNA was digested with BamHI and hybridized to the probe outside of the targeting construct. The BamHI restriction fragments derived from WT and the knock-in alleles are indicated.