Construction and identification of lentiviriral vector over-expressing miR-15a/16-1

Objective:To construct a lentiviral vector that over-expression miR-15a/16-1,and to proceed with the lentivirus package,identification,titer determination and transfection to target cells.Method:miR-15a/16-1sequence was obtained by PCR,and it was inserted into the lentiviral vector LV3,and then it was identified by double-enzyme cleavage and gene sequence analysis and extracted.The recombinant plasmid and packaging plasmid pGag/Pol,pRev,and pVSV-G were co-transfected into 293Tcells with the application of liposomes to package the lentivirus.The viral suspension was collected and diluted with gradient,then it was transfected to 293Tcells and the titre was determined,and last obtained lentivirus was transfected to target cells.Result:Restriction enzyme digestion and sequencing results showed that recombinant plasmid has been successfully constructed and packaged to lentivirus.The viral titer was 1×109 TU/ml in experimental group,and 2×109 TU/ml in control group.The target cells were successfully transfected and the transfection efficiency could reach to 80%.Conclusion:It was successful to construct lentiviral vector of miR-15a/16-1and to obtain efficient lentiviral particle of miR-15a/16-1.