Expression Of ZO-1in IPS Cells Mediated By Lentiviral Vector

Objective:The fundamental researches of cardiovascular disease verified that intercalated disc-related gene ZO-lvaris in different myocardial pathologic conditions,and it interacts with Cx43. This research is about to construct recombinant lentivirus over-expressing vector of porcine ZO-1gene,to infect iPS cells,to obtain iPS cells carrying this target gene.Methods:The ZO-1gene sequence publicated on Gendbank was analyzed,and designed a couple of primer containing both BamHI and SaiI. The porcine ZO-1gene was amplified by PCR, and cloned into lentiviral expression vector, to construct the recombinant Lenti-EF1α-EGFP-TRE-ZO1vector, the vector was confirmed by DNA sequencing. The production of lentivirus were obtained by using pVSVG, delta8.91n and fugene to contransfect293T cells. The titer of lentivirus was testd with fluorescent expression,to infect iPS cells.Results:The recombinant lentivirus plasmid carrying ZO-1was correctly constructed, it was identified correct by double enzyme digestion and DNA sequencing.The lentiviral packaging was succeed, and the titer of lentivirus was1.42*108IU/ml.Conclusion:Lenti-EF1α-EGFP-TRE-ZO1lentiviral vector has been successfully constructed. The titer of lentivirus was1.42*108IU/ml,it was used to infect iPS cells, which will be useful for further study in vivo, to understand its function and its unique effects under different pathological status of heart,and open new doors for the treatment of cardiovascular diseases.