Fig. 1. Generation of human CD4 (CD4) knock-in ES cells. (A) Targeting strategy used to insert CD4 gene into the Scl locus is shown. The mouse Scl locus, targeting construct, and the targeted allele are shown. The black boxes and numbers below indicate the exons. The ATG codon starts within exon IV. (B) Southern blot analysis of the targeted allele. DNA was digested with enzymes indicated and run on an agarose gel. Top left, genomic DNA was digested with EcoRI and probed with genomic DNA as indicated below. Both targeted and wild-type alleles generated a 5.2 kb DNA band. Top right: genomic DNA was digested with EcoRI and probed with exon 6 probe as indicated below. The wild-type allele (upper band) and the targeted allele (lower band) are shown. Bottom: genomic DNA was digested with MunI and probed with human CD4 gene. Only the targeted allele gave 7.3 and 3 kb DNA bands. The gel was run for 48-72 hours for good separation of DNA. The enzymes used are as follows: X, XhoI; R, EcoRI; N, NotI; B, BamHI; H, HindIII; M, MunI.