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. 2020 Dec 23;11:617492. doi: 10.3389/fphys.2020.617492

FIGURE 1.

FIGURE 1

Targeted mutation of MCU using TALENs. (A) Schematic representation of partial MCU genomic sequence demonstrating the TALEN binding sites. Open and solid boxes represent UTRs and coding exons, respectively. (B) Representative genotyping results. Amplification of the genomic region surrounding the deletion using a primer pair FP/RP shown in (A). The wildtype (152 bp) and mutant (141 bp) fragments were separated using polyacrylamide gel electrophoresis. (C) Sequencing analysis of cDNA identified a frameshift mutation caused by an 11-nucleotide deletion in exon 1, resulting in a premature termination of translation. (D) In situ hybridization detects MCU transcripts in the heart (arrow) in WT but not mcu mutant embryos at 3 days post fertilization (dpf). (E) Quantitative PCR analysis of MCU transcripts in 3 dpf embryos. (F) Quantification of Rhod 2 Signals in WT cardiomyocytes with and without MCU inhibitor Ru360 and MCU mutant cardiomyocytes. ****p < 0.0001; ***p < 0.001; and NS: p > 0.05.