Comparision of the Efficiency of Different Promoters Driving the Expression of CD19-specife Chimeric Antigen Receptor (CAR)-redirected T Lymphocytes Mediated by Lentiviral Vector / 医学研究杂志

ABSTRACT

Objective Three kinds of different internal promoters were inserted into the lentiviral vector to drive the expression of CD19-specifc chimeric antigen receptor (CAR)-redirected T lymphocytes with the green fluorescent protein(GFP) and the GFP expression efficiency of transduced cells driven by lentivirus were compared.Methods Three GFP reporter lentivirus vectors carrying different promoters and anti-CD19-CAR,including CMV,EF1α,and CMV-EF1α were selected.Three different vectors' names abbreviate to pHAGE-CMV-EFlα-EGFP-CD19,pHAGE-CMV-EGFP-CD19,pHAGE-EF1α-EGFP-CD19.Human embroic kideny 293T cells were cotransfected with the three plasmids by calcium phosphate DNA precipation.And the expression of GFP was observed under fluorescent microscope after transfecting 293T cells,and virus supernatant was collected after 72 h and centrifuged.Nucleic acid copy number in RNA and the expression of EGFP and CD3 zeta were identified through fluorescence quantitative PCR,flow cytometry and western blotting,respectively.The titers of the lentiviral vectors were determinde by scoring GFP expression following swerial dilutions of the viral supernatant on 293T cells.T lymphocytes was infected with lentivims produced from these reporter vectors.Then,fluorescence microscope,wesyern blotting were used to detect the GFP expression strength.Results Results of fluorescence microscope maintained that different internal promotors driving the expressing effect is different.293T as targeted cell,the transfected 293T cells were found containing strong expression of GFP.The amount of the 293T cells expressing GFP driven by CMV-EF1α promoter was the largest,while the EF1α promoter was the smallest.Flow cytometry results show that the rate of transduction is 72.4％,20.6％ and 14.5％,respectively.T lymphocytes as targeted cell,Western blot showed that CMV-EF1α was the largest among all promoters,while the CMV was the smallest.Conclusion In the study of gene expression mediated by lenuvirus,proper cell lines and promoters should be selected to obtain high efficiency.