Knock out of Fzd9 gene. (A) The restriction map of the locus surrounding the single exon of the Fzd9 gene was used to generate the targeting vector for homologous recombination. The Xs indicate sites of recombination in ES cells that are selected by neomycin (positive selection) and G418 (negative selection against TK). In the mutant chromosome, the Fzd9 gene is replaced by the neomycin-resistance (NEO) gene that introduces a new ScaI site. (B) Southern blot of ScaI-digested DNA from a wild-type (+/+), Fzd9 knock-out (-/-), and heterozygous (+/-) animal hybridized with the 3′ probe indicated in panel A. The 6-kb ScaI fragment is derived from the wild-type allele and the 2.5-kb fragment, from the mutant (KO) allele. (C) Northern blot of muscle and testis RNA from wild-type (+/+), Fzd9 knock-out (-/-), and heterozygous (+/-) animals hybridized with an Fzd9 cDNA probe and a β-actin probe as control for RNA loading. The Fzd9-specific 2.4-kb transcript is absent in homozygous mutant animals and reduced in the heterozygote.