Molecular characterization of myosin light chain 2 gene (mlc2f) and recapitulation of its expression pattern using mlc2f promoter-driven transgenesis in mud loach (Misgurnus mizolepis)

The fast skeletal myosin light chain2 gene (mlc2f) was isolated and characterized from mud loach (Misgurnus mizolepis). The mRNA expression of endogenous mlc2f in wild type adult mud loach was mainly observed in skeletal muscle tissue and much lower levels in skin and gill tissue. A transgenic construct using mlc2f promoter attached to cyan fluorescent protein gene was used to drive skeletal muscle-specific expression of the fluorescent reporter in transgenic mud loach . All transgenic founders exhibited mosaic expression of transgene. A founder transgenic male successfully transmitted the transgene to the subsequent generation. Transgenic CFP signals were not found during embryonic developmental stage but afterward the signals were found to be visualized at 12 hours post hatching. During early larval growth, CFP signals were observed in fin muscles, head muscles and skeletal muscles. At juvenile stage, CFP signals were observed reliably in skeletal muscle. In adult stage of F1 generation, strongest CFP expression was visible in skeletal muscle tissue in accordance with the highest mRNA expression of endogenous mlc2f in muscle. Fluorescent transgene was passed on to the subsequent generation (F2) following the Mendelian single gene inheritance pattern. Histological analysis using F2 transgenic also showed the skeletal muscle-dominant transgenic expression. This study provided evidence of the potential of mlc2f promoter to drive skeletal muscle specific expression in the mud loach. Using mlc2f promoter and CFP as reporter gene, it is possible to monitor development of fast skeletal muscle in real time fashion and on site from larva until adult stage of transgenic mud loach