Construction Of Human Connexins Gene Eukaryotic Expression Vector And Establishment Of Its Stable Transfection Cell Strains

Objective:Gap junction channels regulate the passage of ions and biological moleculesbetween adjacent cells and, therefore, are critically important in the development ofdiseases. The gating of gap junction channels is regulated by the membrane voltage,intracellular microenvironment, interaction with other proteins, and phosphorylation.Each connexin channel has its own property for conductance and molecularpermeability. Our research have proved that the remodling of gap junction had beenoccurred in cerebral vasospasm of Subarachnoid hemorrhage, particularly,theincreasing of Cx43,and the decreasing of Cx40, with cx37and cx45slightly rising.This study is aiming to construct a ideal eukaryotic expression vector, expressingconnexin stably in gap junction mediated intercellular communiction-incompetentHeLa cell lines, which not only established a homotypic/heterotypic gap junction cellmodel for probe the connexin-specific permeability and selectivity efficiently, butalso lay the fundation for the further research into the mechanism of drug targetsbased on connexins and relative signals pathway.Methods：1.The open reading frame sequence of Cx37,Cx40were amplified from the totalRNA isolated from human293T,HUVEC(human umbilical cord venous endothelialcells) cells by RT-PCR,Cx45was amplified from the plasmid of M51.We clonedthe PCR sequence into pIRES2-EGFP plasmid, through restriction enzyme digestion,T4ligase,transformation,and then sequenceing respectively.2. A large-scale plasmid preparation was made to transfection from a singlecolony,We transfected the HeLa cells with the plasmid phCx37-IRES2-EGFP,phCx40-IRES2-EGFP,phCx45-IRES2-EGFP by lipofectamine2000,phCx43-IRES2-DsRed by FuGENE HD in6-well dishes,After2days,transfected cells were eitherprocessed for RT-PCR or immunoblotting (for transient transfection) or fed with800μg/ml G418-supplemented media in24-well dishes. Selection was continued for2 weeks, with medium changes every3–4days, until colonies with stable growth wereobtained. Untransfected HeLa cells were also treated with puromycin and did notsurvive after two weeks. Bulk selection was performed in96-well dishes for5-6weeks after trypsinization of all colonies.3. For later identification,we used RT-PCR and western blot to analysed theexpresion of connexins.Results：1. The eukaryotic expression plasmid pIRES2-EGFP carrying the coding seq-uences of human Cx37, Cx40, Cx45was successfully constructed by sequencing.2. The expression of Cx37, Cx40,Cx43and Cx45in stable transfection of celllines was successfuly analysed by RT-PCR,fluorescence microscopy and Westernblot.Conclusion：The recombinant plasmid phCx37-IRES2-EGFP, phCx40-IRES2-EGFP,phCx45-IRES2-EGFP and the HeLa stable cells strains were achieved successfully,which not only established a homotypic/heterotypic gap junction cell model for probethe connexin-specific permeability and selectivity efficiently, but also lay thefundation further for the research into the mechanism of drug targets based onconnexins and relative signals pathway in vitro.