Generation of the chchd10P83L/P83L zebrafish line. A, Human CHCHD10 and zebrafish chchd10 gene structures and location the ALS-associated P80L variant and orthologous P83L variant in our zebrafish mutant. B, Electropherograms of wild type and homozygous chchd10P83L sequences. The asterisk represents a silent mutation included in our donor template for disruption of the two taqII restriction sites. C, Representative immunoblot of Chchd10 and Chchd2 in whole larval lysate from both wild type and chchd10P83L/P83L larvae. Sdhb was used as a loading control and for quantification. Chchd10 protein levels in whole larvae lysate was significantly reduced in our chchd10P83L/P83L model compared to wild type (t (4) = 4.73, p < 0.01) (Di). A tendency for reduced Chchd2 protein concentration was also observed in our mutant model, though this was not found to be significant (t (4) = 2.094, p = 0.10) (n = 3 per genotype, 5 dpf data represent mean ± S.E.M.) (Dii). Ei, Eii, RT-qPCR of the chchd10 and chchd2 transcripts in chchd10P83L/P83L and wild type larvae. No difference was observed in either chchd10 (t (4) = 1.182, p = 0.30) or chchd2 (t (4) = 0.48, p = 0.66) transcript expression levels (n = 3 per genotype, 5 dpf data represent mean ± S.E.M.).
|
