<p>Chen et al. Supplemental Material</p><p>Supplementary Figure 1. Generation of a SknE2 allele by gene targeting</p><p>Schematic diagram showing the Skn genomic locus, the targeting vector and the mutant allele. The top line shows a partial restriction map of the Skn genomic locus. The Skn genomic locus consists of eleven relatively small exons (E1-E11). The second exon (E2) contains the translation start ATG. The regions between the dotted lines represent the 5’ and 3’ regions of homology respectively and X indicates events of homologous recombination. The location of the fragments used as the 5’ or 3’ external probes in </p><p>Southern blotting are shown, as well as the sizes of the fragments detected for wild-type and targeted alleles. Mating of heterozygous Skn animals to a Cre deleter strain (e.g. - actin::Cre) will simultaneously remove exon 2 and PGK-neo-PA cassette to generate the </p><p>SknE2 allele.</p><p>Supplementary Figure 2. Generation of a SknE9 allele by gene targeting</p><p>Schematic diagram showing the Skn genomic locus, the targeting vector and the mutant allele. The top line shows a partial restriction map of the Skn genomic locus. The Skn genomic locus consists of eleven relatively small exons (E1-E11). The ninth exon (E9) contains the putative active sites for acyltransferase. The regions between the dotted lines represent the 5’ and 3’ regions of homology respectively and X indicates events of homologous recombination. The location of the fragments used as the 5’ or 3’ external probes in Southern blotting are shown, as well as the sizes of the fragments detected for wild-type and targeted alleles. Mating of heterozygous Skn animals to a Cre deleter strain (e.g. -actin::Cre) will simultaneously remove exon 9 and PGK-neo-PA cassette to generate the SknE9 allele.</p><p>Supplementary Figure 3. Generation of a ShhC25S allele by gene targeting</p><p>Schematic diagram showing the Shh genomic locus, the targeting vector and the mutant allele. The top line shows a partial restriction map of the Shh genomic locus. The Shh genomic locus consists of three exons (E1-E3). The first exon (E1) contains the translation start ATG. The regions between the dotted lines represent the 5’ and 3’ regions of homology respectively and X indicates events of homologous recombination. </p><p>The location of the fragments used as the 5’ or 3’ external probes in Southern blotting are shown, as well as the sizes of the fragments detected for wild-type and targeted alleles. </p><p>The presence of the loxP and FRT sites in intron 1 does not appear to affect Shh expression (Figure 2 and data not shown) and mice with or without the PGK-neo-pA cassette in the genome exhibit identical phenotypes. </p>