Deletion of exon 3 of <i>Lmx1a</i> in <i>bsd</i> mutants. A

<p>, PCR amplification of genomic DNA from +/+, +/<i>bsd</i> and <i>bsd</i>/<i>bsd</i> mice with primers specific to each exon of <i>Lmx1a</i> gene. One single band is amplified for each exon and sequenced for all <i>bsd</i> genotypes with no differences between mutants (M) and controls (wt, het) except for exon 3, where no band was amplified in <i>bsd</i> mutants (asterisk). <b>B</b>, Partial traces and sequence of <i>Lmx1a</i> intron 4–5 are shown. At 30 base pairs downstream of exon 4 we identified a single nucleotide polymorphism (SNP)-from A to G- that we used to genotype <i>bsd</i> mice. <b>C</b>, PCR amplification of <i>bsd</i> cDNA from +/+, +/<i>bsd</i> and <i>bsd</i>/<i>bsd</i> mice at E10.5 with primers specific to transcript Lmx1a-001 covering exons 2–8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051065#pone.0051065.s004" target="_blank">Table S2</a>). Bands were amplified for controls (wt, het) but no band was detected in mutants (M). We designed primers for small fragments of the transcript (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051065#pone.0051065.s004" target="_blank">Table S2</a>). Controls (+/+) showed bands for all combinations of primers (numbers on top indicate position of primers forward and reverse, respectively) whereas in <i>bsd/bsd</i> no bands were amplified by primers 13 and 35 (black asterisks). Interestingly, we found a small band of less than 100 bps with primers 24. This band is likely to correspond to the size of the amplicon (322 bp) minus the deleted exon 3 (233 bp) of <i>bsd</i> mutants. <b>D</b>, Diagram showing structure of <i>Lmx1a</i> gene in +/+, <i>mtl</i>/<i>mtl</i> and <i>bsd</i>/<i>bsd</i>. Initiation codon in exon 1 and termination codon in exon 8 are indicated. Point mutation in <i>mtl</i> homozygotes is indicated by a red asterisk and deletion in <i>bsd</i> mutants is shown by a red dotted line. <i>Mtl</i> mutation affects the homeodomain whereas in <i>bsd</i> the deletion of exon 3 involves the LIM2 domain.