Identification of a heterozygous <i>Chd7</i> deletion mutation (<i>Chd7^Ome/+</i>) in circling mice.

<p>(<b>A</b>) Quantitative PCR analysis of <i>Chd7</i> exons 1, 2, 3, 4, 6, 18, and 38 on chromosome 4 using genomic DNA isolated from wild-type (WT1 and WT2) and heterozygous mutant mice (HET1 and HET2). Genomic regions corresponding to the <i>Spag9</i> (on Chr 11) gene and the <i>Rlbp1l1</i> gene (also known as <i>Clvs1</i>; located on Chr 4, downstream of <i>Chd7</i>) were also amplified and serve as additional controls. Data were normalized to <i>Spag9</i> and the band for this gene was assigned the value of 1.0 (<i>y</i> axis). (<b>B</b>) qPCR analysis of cDNA generated from wild-type and <i>Chd7</i> mutant mouse brain RNA. PCR primers anchored in exons 1 and 4 (1F/4R) yield a positive result only for cDNA from heterozygous mutant mice. This product fails to amplify from wild-type cDNA because of its large size (∼2 kb). As expected for a heterozygous deletion mutation, PCR primers anchored in <i>Chd7</i> exons 1 and 2 (1F/2R) yield amplicons from both wild-type and mutant samples. “Neg” indicates a non-template control sample. A 100-bp standard was run in the lane near the right edge of the gel. Asterisks indicate bands that were isolated for sequencing in C. (<b>C</b>) Partial cDNA sequence of the bands isolated in panel B verifies that the mRNA transcribed from the mutant allele results from aberrant splicing of exons 1 and 4. The translation initiation codon is indicated in exon 2 of the wild-type allele, and has been deleted from the mutant allele.