GREEN FLUORESCENT PROTEIN MARKS SKELETAL-MUSCLE IN MURINE CELL-LINES AND ZEBRAFISH

The green fluorescent protein (GFP) acts as a vital dye upon the absor ption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the livi ng organism. We inserted the cDNA encoding this 238-amino-acid (aa) je llyfish protein into an expression vector containing the rat myosin li ght-chain enhancer (MLC-GFP) to evaluate its ability to serve as a mus cle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously thro ughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibrob lasts transfected with MLC-GFP did not fluoresce, When GFP and beta Ga l markers were compared, the GFP signal was visible in the cytoplasm o f the living cell, whereas visualization of beta Gal required fixation and resulted in deformation of the cells, When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression wa s apparent within 24 h of development. gfp expression was never observ ed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 mont hs, demonstrating that GFP is an effective marker of muscle cells in v ivo.