Construction and identification of RNAi lentiviral vector on murine RelB / 解放军医学杂志

ABSTRACT

Objective To construct a lentiviral expression vector of murine RelB for RNAi,and then to effectively silence the RelB gene expression of murine bone marrow derived dendritic cells for constructing the bone marrow tolerogenic dendritic cell,in order to provide an experimental foundation for a novel clinical therapy of autoimmune disease.Methods RelB shRNA sequence of mouse was designed by on-line designer software on Corp.Invitrogen,after synthesis and annealing,double strand oligonucleotides(dsoligoes)were cloned into the pENTRTM/U6 plasmid,then after sequencing,a positive clone was further subcloned into pLenti6/BLOCK-iTTM-DEST vector.It was then transformed into stb13 competent cell,and after sequencing,293FT cell line was transfected by above positive recombined plasmid and lentiviral packing materials.It was incubated for 48 to 72 h in a 37℃,5% CO2 incubator.Culture supernatant was harvested and stored at-80℃,then the virus titer was determined by serial dilution assay.Results It was showed from sequencing figures that all the pENTRTM/U6-RelB-shRNA plasmids were positive clone vector,and this positive recombinant vector was recombined with pLenti6/BLOCK-iTTM/U6-DEST vector.It was then transformed into stb13 competent cell and screen positive clone by ampicillin,and resequenced by the use of primer forward U6 primer.The results also showed that recombinant lentiviral vector was positive clone.Viral particle was packaged with other packaging material mediated by lipofectamine 2000 in 293FT cell line.Cultural supernatant was collected and stored at-80℃,and lentiviral particle titer was determined by serial dilution assay with 6?105/transduced unit.Conclusion Lentiviral shRNA expression vector of murine RelB gene for RNAi was successfully constructed.It might be a rational procedure to make tolerogenic DC,and then to develop a DC vaccine and DC-based immunotherapy for autoimmune diseases.