Construction and Identification of Lentiviral Vector Carrying Rat Smad7 Gene

Objective:To construct the lentiviral vector carrying rat Smad7 gene.Methods:Smad7 gene sequence was amplified by RT-PCR from extraction of total RNA of rat cerebral cortex and kidney.The Smad7 gene was cloned into the lentiviral transfer vector,pCDH-CMV-MCS-EF1-copGFP,to acquire the recombinant plasmid which contained Smad7 and green fluorescent protein(GFP) reporter.After transformation reaction,candidate clone was further analyzed by PCR and gene sequencing.Virus particles were collected after the recombinant Smad7 plasmid transfection with lentivirus package plasmids mix.Viral titers were examined by transduction of H1299 cells with serially diluted virus supernatants followed by cytofluorimetric analysis of GFP positive cells.Results:The recombinant lentiviral transfer vector plasmid was identified to be correct by double restriction enzymes,and the cloned Smad7 gene sequence was consistent with the sequence reported in Genebank.The lentiviral supernatant was collected and concentrated,and the titer of lentiviral vector particles was found to be 1.0×107ifu/ml.Conclusion:The lentiviral vector carrying Smad7 gene has been constructed successfully.