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Fig. 3. Hypomorphic expression of the Dkk1^d (doubleridge) allele detected by primer extension and wholemount in situ hybridization. (A) Quantitative assay of allelic Dkk1 transcripts in heterozygous mice. Primers 1 and 2 are used for RT-PCR amplification of transcripts, and the 25 bp primer 3 for primer extension of the amplified RT-PCR product. The single nucleotide polymorphism in the 3' UTR that distinguishes the C3H and SJL transcripts is shown in bold. The extension products obtained in the presence of ³³P-ddGTP are 31 bp in length for the C3H allele and 33 bp in length from the SJL allele, the parental allele for the doubleridge insertion. (B) Primer extension products are separated on a 10% acrylamide gel and visualized using BIOMAX film. Triplicate assays of four RNA samples from pooled hindlimbs at E13.5. Samples 1 and 2, (C3H X SJL)F1-Dkk1^d/+ doubleridge heterozygotes; 3 and 4, (C3H X SJL)F1 wild-type heterozygotes. (C-F) Wholemount in situ hybridization of Dkk1^d/d embryos and +/+ littermates with a Dkk1 cDNA probe demonstrates reduced expression in multiple domains. (C,D) E9.5 forelimb; (E,F) E9.5 tail bud.

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