Fig. 1
NebY2303H,Y935X mice express 50% of the Tyr2303His missense allele, while the Tyr935* nonsense transcript is not detected. a A schematic representation of the location of the selected mutations on the nebulin protein. Primers, which differentiate between the (b) wild type (WT; Tyr2303, T allele) and (c) missense (MUT; Tyr2303His, C allele) transcripts were used to generate allele-specific qPCRs. Relative Neb expression was determined using the delta Ct method and normalised to the geometric mean of two endogenous control genes, Tbp and Eef2. Expression of Neb transcript from NebY2303H,Y935X mice was compared with expression from the parental lines (NebY2303H(+/+), NebY2303H(+/−) and NebY935X(+/−)) and the C57BL/6J background strain. As expected, NebY2303H,Y935X mice expressed approximately 50% of the mutant allele p.Tyr2303His compared with homozygous NebY2303H(+/+) mice, and no clear expression was detected of the WT Tyr2303 allele, supporting the hypothesis that the Tyr935* transcript (carrying the Tyr2303 WT allele) is lost due to nonsense-mediate mRNA decay. The low level of WT transcript detected in NebY2303H,Y935X and NebY2303H(+/+) samples is likely due to background amplification from the mutant allele since the WT and mutant alleles differ by only one base-pair. d Representative image of the protein analysis on a 1% SDS agarose gel across the mouse strains, and e quantification of nebulin protein, normalised to myosin heavy chain (MyHC). Nebulin protein levels remained comparable across genotypes, suggesting a compensational mechanism in the NebY2303H,Y935X and NebY935X(+/−) mice from the transcript not undergoing nonsense-mediated decay. Unpaired Mann-Whitney, n = 3, ns, p > 0.05