We replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette [21] or an IRES-HygR cassette by promoter trap selection. We amplified the 5' arm (1.8 kbp) using KOD plus (TOYOBO) with the pH34-targetpair5-U (5'-CCGCGGAAAGTCAAGAGATTGGGTGG-3') and pH34-targetpair5-L (5'-GCGGCCGCCTTTACGGGTCACGAGGGTCAC-3') primers. The 3' arm (5.8 kbp) was amplified using the pH34-targetpair3-U (5'-TGTGGCCAGTGTTTGGTTCTGGCGGG-3') and pH34-targetpair3-L (5'-CTCGAGGACTCGCCATTCTAGCCAAG-3') primers. The IRES β-geo or IRES HygR cassettes were ligated in between the two PCR fragments. The diphtheria toxin A cassette was placed downstream of the 3' arm. After linearization with SacII, these targeting vectors were electroporated into 2.0 × 107 RF8 ES cells [22] using a Gene pulser (BIORAD). Transfected cells were selected with 250 μg/mL G418 or 100 μg/mL hygromycin B, respectively. Genomic DNA from G418- or hygromycin B-resistant colonies was screened for homologous recombination by Southern blotting.