Generation of mES cells homozygous for the Socs3 knock-out allele by high-dose puromycin selection. (A) Strategy for the generation of Socs3-KO ES cells. The schematic maps of the Socs3 allele (top), the KO vector carrying the Cherry-IRES-pac-pA cassette (middle), and the KO allele generated by homologous recombination (bottom). The PCR with the primers set at the 5′ external genome and the open reading frame (ORF) in the second exon provides the polymorphism between the wild-type and mutant alleles, 2.1 kb and 4.8 kb, respectively. (B) Sensitivity of the heterozygous clone to various concentration of puromycin. Most of the cells were killed by 3.0 μg/ml of puromycin for 5 days. (C) PCR analysis of the genotypes of the clones selected by 6 μg/ml of puromycin from the heterozygotes. All clones possess the mutant allele only, indicating that they are homozygotes for the Socs3-KO allele.