Construction and identification of miR-186 overexpression lentiviral vector / 吉林大学学报(医学版)

ABSTRACT

Objective: To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and to explore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods: The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoR I and Age I restriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results: The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU · mL-1 and 5 × 108 TU · mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12. 640 0 ± 0. 788 4) was significantly increased by 15. 07 times (t=14. 72, P<0. 01) compared with control group (0. 838 7 ± 0. 145 6). Conclusion: The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus successfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.