Cloning of Sheep FNDC5 Gene into Non-Viral Vector

Nowadays the importance of large fat-tail in sheep has reduced and reduction in fat-tail size is most favorable for breeders and consumers. In this regard, FNDC5 gene is a candidate gene that is effective on reducing fat via stimulating white-tobrown fat conversion by secretion of a membrane protein that is called Irisin, which induce uncoupling protein 1 productionin brown fat. Brown fat cells due to possess of large numbers of mitochondria that contain UCP1, causes that, the energy inthe mitochondrial released in the form of heat rather than being stored as fat. Thus, the cloning of FNDC5 gene with anexpression specific promoter in fat-tail tissue of sheep could be a suitable strategy for reducing fat-tail storage through genetransfer in subsequent studies. Therefore, in this research, the mRNA was extracted from heart tissue of sheep and used for cDNA of FNDC5 gene construct. However, due to lack of appropriate expression of the target gene, we did not succeed to construct the FNDC5 cDNA by mRNA extracted. Hence, the FNDC5 CDs sequence was synthesized, so that, restrictionsites of EcoR1 and Xho1, corresponding restriction enzyme sites in pIRES-hrGFP vector, was designed at the end offragment. In the next step, ligation between the vector and the fragment was performed via T4 ligase enzyme and thereupontransformed to DH5α competent cells. Then, presence of the target gene in the recombinant pIRES-hrGFP-FNDC5 vectorwas verified using plasmid isolation, enzymatic digestion and PCR. In conclusion, our results showed that cloning of FNDC5 gene in pIRES-hrGFP vector successfully performed and it's prepare for following studies.

Keywords:

FNDC5 gene , Cloning , pIRES-hrGFP , sheep