Construction and identification of human eukaryotic expression vector of hsa-miR-134

Abstract:

Objective：To construct the eukaryotic expression vector containing exogenous miR-134 precursor，to study its expression in the human oligodendroglia（OL） cells and to provide the technical platform for further studying the function of miR-134 and its target gene. Methods：miR-134 precursor sequence was amplified from the human genomic DNA and cloned into pEGFP-N1. After being confirmed by PCR and sequencing，recombinant plasmid was transfected into human OL cells and transcription level of miR-134 was detected by real-time PCR. In addition，transcription efficiency was monitored by the inverted fluorescence microscopy. Results：The target bands were consistent with the expected results by enzyme digestion and PCR identification，and the inserted sequence was correct in gene sequencing detection. After the recombinant plasmid being transfected into OL cells，fluorescence quantitative PCR detection showed that there was no difference between pEGFP-N1 empty plasmid group and control group in the expression level of miR-134（P=0.882）. Moreover，the miR-134 expression level of pEGFP-miR-134 plasmid transfected group was 52 times over that of pEGFP-N1 empty plasmid group，with significant statistical differences（P=0.023）. Conclusions：The miR-134 expression vector is successfully constructed，which paves the way for the future experiment.