Construction Of Lentiviral Vectors Targeting C57BL Mouse Survivin Gene By RNA Interference And The Effect On Dendritic Cells In Vitro

Background and Objective:Rejection after renal transplantation not only bring a tremendous financial burden to patients, But also can result in kidney lost of patients in Serious situation. In addition to anti-rejection drug treatment, There is no effective treatment at present. At present,It is very important To explore a treatment to reduce or even eliminate rejection after renal transplantation. In this study, lentiviral virus of low toxicity is used to construct lentiviral vectors targeting mouse survivin gene by RNA interference,Then study the infection efficiency and apoptosis index on bone marrow-derived immature dendritic cells in vitro, This approach lays the foundations for the late experiments .Methods:The effective sequence of siRNA targeting survivin gene was confirmed in our previous experiment.The complementary DNA containing both sense and antisense oligonucleotides of the targeting sequence was designed synthesized.After being annealed , the double-stranded DNA was inserted into the pLVTHM vector.293T cells were cotransfected with PLV- shRNA,pRsv-REV, pMDlg-pRRE, pMD2G ,the titer of virus was tested according to the expression level of GFP.The recombinant lentiviruses were used to infect DC separated from the bone marrow of mice in vitro.The infection efficiency was assessed by fluorescence microscope. The flow cytometry was used to detect the apoptosis index of immature dendritic cells in vitro.Results:according to enzyme cutting and sequencing, the lentiviral vectors targeting C57BL mouse survivin gene by RNA interference was constructed successfully,which had a high transfection efficiency and low apoptosis index.Conclusion:In this study, lentiviral virus of low toxicity targeting mouse survivin gene by RNA interference was successfully constructed.It had a high transfection efficiency and apoptosis index,which laid the foundations for the late experiments.