Construction of Porcine microRNA-124 Overexpression Vector and Establishing of Stable Transfected Cell lines

microRNA-124(miR-124),high expression in brain,regulate neuronal differentiation and the pro- liferation of cancer cells. The porcine miR-124 precursor is transcribed from chromosome 4 and 14. In this study,the miR-124 precursor was amplified by PCR from landrace genomic DNA,ligated into T vector,then digested by EcoR Ⅰand NotⅠ,the digested fragment were connected into PiggyBac(PB) transposon vector,then the recombinant vector(PB-miR-124a1,PB-miR-124a2) was transfected to procine kidney epithelial cells(PK15). The transfection efficiency was detected by expression of green fluorescent protein. The stable trans- fection cells were screened by puromycin,and the expression of miR-124 was determined by qRT-PCR. The results showed that the PB transposon recombinant vector(PB-miR-124a1,PB-miR-124a2) were constructed successfully with high transfection efficiency. The best stability screening concentration of puromycin was 2 μg/mL. qRT-PCR results showed that the expression of miR-124 in transfected PB-miR-124a2 group was increased significantly compared with control groups(P0.01),but in transfected PB-miR-124a1 group,the expression of miR-124 increased not significantly(P=0.06). These results demonstrated that the basal level of expression of miR-124 mainly transcribed from ssc-mir-124a2 gene. The study provides a foundation material for porcine miR-124 function research.