FIG. 1

Gene targeting of the murine 11-cis-RoDH gene. (A) Genomic structures of the murine 11-cis-RoDH (roman numbers) and GCN5L1 (arabic numbers) genes (upper panel), structure of the targeting vector (middle panel), and genomic structure of the targeted 11-cis-RoDH gene (lower panel). Homologous recombination results in the replacement of exons I, II, and III of the 11-cis-RoDH gene by a neomycin cassette (NEO). Positions of the 5′ and 3′ probes are indicated. Asterisks mark positions of the primers used for PCR genotype analysis. Abbreviations: B, BamHI; E, EcoRI; K, KpnI; N, NheI; S, SstI; HSVtk, herpes simplex virus thymidine kinase gene. (B) Southern blot analysis of the cell lines. Autoradiograms of three Southern blots containing genomic DNA from three targeted ES cell lines and from mouse strain 129/Sv after hybridization with the 5′ probe (left), 3′ probe (middle), or neo probe (right). Sizes are shown in kilobases. (C) PCR genotyping of mouse tail DNA. PCR fragments derived from wild-type (11-cis-RoDH^+/+), heterozygous (11-cis-RoDH^+/−), and homozygous (11-cis-RoDH^−/−) mice. Offspring were analyzed with primers KORDH-s1 and neo-a1. The presence of a targeted allele results in the amplification of a 365-bp fragment (left panel). Offspring from 11-cis-RoDH^+/− parents were analyzed with primers KORDH-s1 and KORDH-a1, which results in fragments of 2.9 kb in 11-cis-RoDH^+/+ mice, 1.5 and 2.9 kb in 11-cis-RoDH^+/− mice, and 1.5 kb in 11-cis-RoDH^−/− mice (right panel).