Molecular Cloning and Motif Identification of the Sheep Musclin Gene Promoter.

Musclin is a bioactive factor that functions in regulating the muscle growth and metabolism. To investigate the transcriptional regulatory mechanism of the gene, the 1.4 kb musclin promoter in sheep was cloned (GenBank accession: JX966391) and the sequence was analyzed to predict the motifs associated with muscle growth. Next the enhanced green fluorescent protein (EGFP) was selected as the reporter gene and various wild-type and motif-mutant vectors were constructed. The transcriptional regulatory activities were compared by observing the fluorescence strength and detecting the EGFP mRNA expression in C2C12 myoblasts transfected with the vectors. The results showed that the different lengths of promoters could drive the transcription of EGFP and the mutation of some motifs up- or downregulated the activity of the promoter. Furthermore, the electrophoresis mobility shift assay showed that these motifs regulated the musclin gene transcription through binding to the corresponding transcriptional factors in sheep muscle tissue.