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KMID : 0381120130350030289
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Genes and Genomics
2013 Volume.35 No. 3 p.289 ~ p.303
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Molecular characterization of fast skeletal muscle-specific myosin light chain 2 gene (mlc2f) in marine medaka Oryzias dancena
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Lee Sang-Yoon
Kim Dong-Soo
Nam Yoon-Kwon
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Abstract
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The genomic structure including the 5′-upstream regulatory region of the fast skeletal myosin light chain 2 gene (mlc2f) was characterized in the marine medaka (Oryzias dancena; Beloniformes). Molecular phylogenic analysis inferred that the marine medaka mlc2f should belong to the teleost mlc2fa group characterized by seven-exon organization. Bioinformatic analysis of the regulatory region represented the various transcription factor binding motifs especially including myogenic regulatory factor binding sites such as myocyte enhancer factor-2 site and E-box. Real-time RT-PCR assays revealed that the mlc2f mRNA was highly predominant in skeletal muscles and also that the muscular mlc2f expression was little modulated by environmental salinity ranging from 0 to 30 ppt. The mlc2f mRNA expression was differentially modulated during embryonic development of this species, in which the expression of mlc2f was stimulated from the retinal pigmentation stage and then markedly activated until hatching. From the microinjection-based introduction of a green fluorescent protein gene (gfp) driven by a 2.95-kb marine medaka mlc2f promoter into the marine medaka embryos, the onset of transgenic GFP expression was in accordance with that of endogenous mlc2f gene. Although all the microinjected embryos and resultant F0 fish showed a mosaic distribution of GFP expression with some ectopic expression pattern, the overall expression of transgenic GFP was enriched in the skeletal muscles. However, transgenic hemizygous F1 fish produced from the germline positive founders showed uniform, fast-skeletal muscle-specific expression of GFP, which resembled the pattern of the endogenous mlc2f gene expression. The expression of transgenic GFP was observable mainly in head region and weakly in caudal peduncle of the hatched larvae. The GFP expression was gradually intensified in these sites and became spread over other skeletal muscle parts with larval ages, such that the fish at 21 days post hatching acquired uniform GFP expression over nearly whole skeletal muscles. Data from this study suggest that the mlc2f promoter-driven transgenesis holds promising potential to monitor the differentiation of the fast skeletal muscles of this species in a real-time fashion and also to drive efficiently the expression of foreign proteins in the marine medaka skeletal muscles.
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KEYWORD
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Marine medaka Oryzias dancena, Microinjection, mlc2f gene and promoter, mRNA expression, Molecular phylogeny, GFP expression
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