The Creation Of A Tissue Specific Expression Connexin 31 Construct

BackgroundConnexins is a protein family that can form a Gap Junction structure on the membrane and allows the exchange of small molecules(＜1kD) between cells.The cells then synchronize their response of foreign stimulus.Connexin31(CX31)is one member of the Connexin family which its mutations are found to cause dominant or recessive EKV,dominant or recessive hearing impairment and dominant hearing impairment combined with peripheral neuropathy.There is no hearing loss or skin disease phenotype found in CX31 knockout mouse and a research which created a transgenic mouse in using of human CX31 promoter found partially keratinise of the epidermis.CX31 plays very important role in cell stability,proliferation and differentiation.But we still know little of CX31 character and activity in Gap Junction assembling,permeability, function regulation and intra-cellular trafficking.Research goalsTo enhance the expression level of CX31,especially some mutation's expression in specific tissue may cause the phenotype in animal models.We employed a Keratin 14 promoter and made an expression construct of CX31 wt and F137L mutant,then studied its expression ability and specificity in skin sourced cell line.We obtain a skin tissue specifically expressed construct which also can be used to create a transgenic animal model.MethodsWe use PCR to amplify CX31 wt and F137L mutant and ligate it into a vector contains Keratin14 promoter,rabbit globin and Keratin 14 3' Untanslated region elements.Thereafter,tansfect the constructs in to Hacat cell use Lipofectamine LTX and detect the CX31 with immunoprecipitation and immunostaining.We also transfect the HEK 293T cell by calcium phosphate method and detect its expression level by immunostaining in order to evaluate the ability of tissue specific expression.Results:The constructs are established by enzyme digestion and sequencing. The expression in Hacat cell is detected by immunoprecipitation and immunostaining,which the CX31 wild type protein can form the Gap Junction plaques while the F137L mutant can not.We do not detect the CX31 expression in HEK 293T cells by using Keratinl4 promoter,but the positive control which was transfected with pCDNA3.1A(-)-CX31-myc has the expression and Gap Junction plaques.Conclusions The two constructs made in this research can successfully and specifically express in skin tissue sourced cell line.They also are available in the creation of transgenic animal model after linearization to remove their vector backbones.