Overexpression of Tmie causes increased bundle expression of Tmc1-GFP and Tmc2b-GFP. (A) Semi-quantitative PCR of tmie cDNA using primers within exon 4 to detect transcripts from both transgene and the endogenous gene. We generated cDNA from RNA extracted from 5 dpf whole larvae. Larvae carrying Tg(myo6b:tmie-p2A-NLS(mCherry)) had 6.5-fold more tmie transcript than wild type non-transgenic siblings. Products are from 40 cycles. Less PCR reaction (2.5x less) was loaded for gapdh to avoid saturation. (B) Confocal images of the lateral cristae in 4 dpf tmieru1000 larvae co-expressing two transgenes, tmc1-GFP (upper panel) and tmie-p2A-NLS(mCherry) (lower panel). The p2A linker is a self-cleaving peptide that results in equimolar translation of Tmie and nuclear mCherry. (C) Plot of the integrated density of Tmc1-GFP fluorescence/crista in the ROI, expressed in arbitrary units; significance determined by one-way ANOVA. (D-E) Same as B-C except using tmc2b-GFP instead of tmc1-GFP. All statistics are mean ± SD. Scale bars are 5μm.
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