Fig. 1. Gene targeting of the murine Wnt11 locus. (A) Targeting strategy. Genomic sequences spanning the Wnt11 locus were cloned and subjected to restriction mapping and sequencing to locate the intron-exon boundaries. The homologous recombination events lead to deletion of exons 4 and 5 and around 1.5 kb of intron 5 and lead to generation of a truncated transcript at amino acid 28 onwards in the corresponding Wnt11 protein. The 5', 3'and neo probes used to screen for gene targeting with Southern blot are indicated, and SpeI digestion was used as a diagnostic enzyme to screen the targeting event. Cutting of the wild-type locus with SpeI was expected to generate around a 20 kb fragment where the PGKneo introduces additional SpeI in the targeted allele and was expected to generate a 10 kb fragment. Primers to monitor the wild-type and mutant allele are also indicated with arrowheads. (B) Genotyping the Wnt11 knock out allele. Genomic DNAs from Wnt11 wild-type, heterozygote and homozygous mutant alleles were digested with AflII, Southern blotted and probed with the Wnt11 cDNA 240 bp kb Nco fragment within Exon VI. The Wnt11 allele specific polymorphism is shown whereby the Wnt11 wild-type allele is associated with a 7.0 kb band, while the Wnt11 mutant allele is associated with a 5.5 kb band. (C) Wnt11 homozygous mutant kidneys produce a shortened Wnt11 mRNA. Genespecific primers were used to RT-PCR wild-type and mutant P1 kidney mRNA. The wild-type product is 1.8 kb, while the Wnt11 mutant product is 1.3 kb, in agreement with the expected size resulting from deletion of Wnt11 exons IV and V. (D) The first 28 amino acids of the mutant Wnt11 protein match the wild-type sequence. Downstream of the exon IV/V deletion (arrow), the reading frame is out of frame resulting in a null allele.