Construction Of The Over-expression Lentivirus Vector Of The Gene GJB6and Its Expression In HaCaT Cells

ObjectiveTo construct the lentiviral expression vector of the pathogenic mutations in gene GJB6encoding connexin-30(Cx-30) and three mutations in Clouston syndrome and to analyse the expression in immortal keratinocytes(HaCaT cell).Materials and MethodsTo construct the over-expression lentivirus vector of the gene GJB6and the mutations. The expression was tested and HaCaT cell was transferred using fluorescent inverted microscope, q-PCR, Western Blot et al.ResultsThe lentiviral expression vector of the gene GJB6was constructed successfully and the HaCaT cell was transferred successfully by two mutations (G11R and A88V).There were many findings as follows in the process of construction and transfection. Strong fluorescence was expressed after the293T cell was transferred by wild type recombinant plasmid and strong positive stripe was showed using Western Blot.Stong fluorescence could be observed in293T cells transferred by A88V and less-fluorescence could be observed in293T cells transferred by G11R after the293T cells were transferred by the recombinant plasmid of three mutations and fluorescence could be observed in the cells transferred by V37E but weakly positive stripe was showed using Western Blot. It declared that target gene was expressed in293T cells but the expression was low comparing the wild type. Less-fluorescence could be observed after HaCaT cells were transferred by the lentiviral expression vector of the mutations (G11R and A88V) and fluorescence could not be observed in the cells transferred by V37E and a mass of HaCaT cells were died.ConclusionProtein could be located on cytoplasmic membrane after HaCaT cells were transferred by wild type GJB6.Only G11R and A88V in three mutations could locate on cytoplasmic membrane but Cx-30encoded by them was less expressed. We presume that it impacted on normal proliferation and growth of the kertinocyte that different mutations in gene GJB6could not locate on cytoplasmic membrane of kertinocyte or could not form functional Cx-30.