Construction And Identification Of Lentiviral Vector Carrying Mouse Proinsulin Gene

Objective: Clone mouse proinsulin(PI) gene fragment by molecular biology process,use pLenti6/V5-D-TOPO vector to create lentivirus expression plasmid . Methods: 1. Extract mouse pancreatic island ,obtain mouse pancreatic island cDNA by RT-PCR,design primer Include the 4 base pair sequences (CACC) on the 5′end of the forward primer,which is necessary for directional insert into vector. the PI open reading frame cDNA was generated by RT-PCR using specific primers .Use agarose gel electrophoresis and DNA sequencing to check the integrity of the PCR product.2.The construction of retrovirus vector,When sequencing is correct, PI gene was conected in to pLenti6/V5-D-TOPO vector following TOPO Cloning reaction.rename the expression vector PI-pLenti6/V5-D-TOPO.Transform One Shot? Stbl3? Chemically Competent E. coli ,Amp selective plate incubate the transformation mix ,after plasmid extraction ,agarose gel electrophoresis and DNA sequencing is performed to confirm the integrity of PI-pLenti6/V5-D-TOPO.Results: 1. Total RNA of islands of pancreas was extracted. PCR amplification of cDNA and PI gene was cloned by RT PCR. We acquired an 378bp DNA fragment including PI gene ORF sequence.The PI ORF sequence is identical with the sequence searched from GeneBank. 2.This fragment was constructed into the PI-pLenti6/V5-D-TOPO lentiviral vector and was confirmed to be the correct sequence of gene by agarose gel electrophoresis and DNA sequencing. the result of sequence analysis was exact.Conclusion: 1.mouse proinsulin gene could be successfully cloned though molecular biology process ,and usegene engineering method construct lentiviral expression plasmid carrying Proinsulin gene.2. The PI-pLenti6/V5-D-TOPO expression plasmid could be used to Cotransfect with the ViraPower Packaging Mix into the 293FT Cell Line to produce lentivirus. Which is based on furth research of the function of PI in curing type 1 diabetes in NOD mouse.