Fig. 2. Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele (Isl1^CKO). Isl1 genomic structure and restriction enzyme map is shown at the top. White boxes are the non-coding exon sequences and black boxes the coding sequences. Thick bars are the sequences used to generate the homologous arms in the targeting vector. (B) Generation of Isl1^lacZ knock-in allele. Neo, PGK-neomycin resistance gene; DTA, diphtheria toxin gene for negative selection of embryonic stem cells; lacZ, β-galactosidase-encoding gene; FRT, flipase recognition sequence; loxP, Cre recombinase recognition sequence; B, BamHI; RI, EcoRI; RV, EcoRV; K, KpnI; N, NotI. (C) Left panel, Southern genotyping confirmation of Isl1^lacZ and Isl1^lox mice using 3' probe and BamHI digestion. Right panel, PCR genotyping using primers indicated in A&B can distinguish Isl1^lox, Isl1^lacZ and wild-type allele. (D) Anti-ISL1 immunolabeling of E13.5 retina sections confirms the deletion of ISL1 in the retina of Isl1^loxP/lacZ; Six3-Cre mice. The enlarged views of the corresponding boxed regions are showed on the right. Arrows indicate residual ISL1 expression. R, retina; L, lens. Scale bars: 50 µm.