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Abstract microRNA-124(miR-124), high expression in brain, regulate neuronal differentiation and the proliferation 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 transfection 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 (P＜0.01), 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.
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Received: 21 June 2013
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