Replacement of the βS-globin gene with a βA-globin gene in knock-in sickle ES cells. (A) Schematic representation of the human β-globin locus, mouse β-globin locus, and the human –1400 ^Aγ-β^S knock-in locus in ES cells derived for this study. Arrows indicate DNase I hypersensitive sites (HSs) that mark the locus control region (LCR). Red and yellow boxes represent functional human and mouse genes, respectively. White boxes represent pseudogenes. Black circles indicate loxP sites. (B) Schematic representation of gene replacement in knock-in sickle ES cells. The 24-kb replacement vector contains a 2.1-kb PGK/TK marker gene, 1.7 kb of mouse 5′ flanking sequence, a –383 γ-β^A fragment (8.7 kb), a 1.8-kb floxed PGK/Hygro gene, and 7 kb of mouse 3′ flanking sequence. Homologous recombinants were identified by PCR with primers 1 and 2 to identify correct 5′ sequences (primer 1 is outside of the vector homology region) and with primers 5 and 6 to identify correct 3′ sequences (primer 6 is outside of the vector homology region). PCR with primers 3 and 4 followed by Bsu36I digestion was used to distinguish β^S and β^A alleles. (C) 5′ PCR (primers 1 and 2) and 3′ PCR (primers 5 and 6) from 2 positive homologous recombinant ES cell lines (clones 1 and 2). In clone 1, recombination at the 5′ end occurred downstream of the –383 γ sequence; therefore, the replacement allele maintained the –1400 γ promoter (2.8-kb PCR product). In clone 2, recombination at the 5′ end occurred upstream of the –383 γ sequence; therefore, the replacement allele contained the –383 ^Aγ promoter (1.8-kb PCR product). Bsu36I digestion of PCR fragments derived with primers 3 and 4 is presented in the second panel of panel C. β^A fragments are digested, but β^S fragments are resistant to digestion.