Construction of a Porcine Skeletal Muscle-Specific Promoter by Inducing the Seed Region of miR-208a.

Abstract

Transgenic promoter systems are of great interest for their potential use in gene therapy or production due to their high activity, long term, and cell specificity. Here, in order to obtain promoters with high activity and expressed specifically in skeletal muscle, the MYOD1, MYF5, and MCK were selected as the candidate genes. The truncated promoters were amplified and their activity was verified through dual-luciferase reporter gene test. We used genetic engineering techniques to improve promoter activity by tandemly linking enhancers and promoters or two promoters. Furthermore, synthetic promoter was the most active when two eMCK enhancers and pMCK promoter were cascaded. To improve the tissue specificity of the promoter, the seed region of translational repressor miR-208a was inserted into the downstream of the promoter (pGL3-2eMCK-pMCK-T208-mCherry-EGFP). The results showed that the expression level of target genes decreased significantly (P < 0.05) in myocardium rather than in skeletal muscle. The results of in vivo transfection indicated that tandem transcriptional regulatory elements can increase promoter activity in mice. This work laid the foundation for future research on genetically modified pigs.