The Model Of HEK293T Co-transfected By Human Scn5a Gene And PRKAG2Gene

BackgroundAdenosine monophosphate-activated protein kinase (AMPK)γ2subunit encodedby the gene PRKAG2.AMPK is a serine/threonine protein kinases.AMPK is presentin various body tissues and cells, including heart.PRKAG2gene mutation causesAMPK dysfunction and a series of heart disease,therefore this type of disease isnamed PRKAG2heart syndrome.It is an autosomal dominant hereditary heartdisease,because PRKAG2gene which encoding AMPK γ2subunits occurrs the pointmutations.Most patient suffers from familial hypertrophic cardiomyopathy, ventricularpre-excitation and myocardial glycogen deposition. Currently,foreign scientists founda total of17PRKAG2heart syndrome families and made genetic analysis for thesefamilies.Foreign scientists found a total of nine kinds of mutations, including one kindof frameshift mutations and eight kinds of missense mutations.All mutations arelocated in cystathionine-β-synthase (CBS) functional area.In2007, Our lab firstreported a China family with PRKAG2heart syndrome.We discovered a new kind ofPRKAG2mutation-glycolamine (100) to serine (G100S).We are interested in thismutation which is located in the area of non-CBS.Accordingly, our laboratory carriedout further research for this new mutation.The purpose is to reveal the pathogenesis ofChinese people PRKAG2heart syndrome.Previous studies have found thatrPRKAG2G100S mutant decreasing AMPK activity.It caused rat cardiomyocyteglycogen deposition and zebrafish myocardial hypertrophy.This finding is consistentwith foreign research results.Thus, we will further research the role of the G100Smutation in PRKAG2cardiac Syndrome arrhythmia.ObjectiveIn this study, the human cardiac sodium channel gene (Scn5a) and the PRKAG2gene were co-transfected into HEK293T cells.In order to research the function of thehuman cardiac sodium channel affected by PRKAG2gene new mutations.Methods1.The over-expression vectors p3×FLAG-CMV-7.1-PRKAG2of the PRKAG2gene wild-type, R302Q mutant and G100S mutant were respectively constructed andtransfected into HEK293T cells;2.The over-expression vector Pcdh-GFP-Scn5a of the Scn5a gene were constructed and transfected into HEK293T cells;3.The over-expression vector Pcdh-GFP-Scn5a and The over-expression vectorsp3×FLAG-CMV-7.1-PRKAG2of the wild-type PRKAG2gene, R302Q mutant andG100S mutant respectively were co-transfected into HEK293T cells;Detecting the result of vector construction by PCR, electrophoresis and DNAsequencing.Detecting the result of co-transfection by real time PCR, western blot andimmunofluorescence.Results1.The PRKAG2gene wild-type, R302Q mutant and G100S mutant over-expressionvector were successfully respectively constructed and transfected into HEK293Tcells.(1)The result of p3×FLAG-CMV-7.1-PRKAG2sequence was consistent with theCDS sequence of human PRKAG2gene.(2)Forty-eight hours after transfected into HEK293T cells, Throughimmunofluorescence staining,we can see the red fluorescence in HEK293T cellsunder a fluorescence microscope;RNA transcript level of the PRKAG2gene wasverified by real-time PCR,RNA transcript level in transfection group wassignificantly higher than that control group;total cellular protein was verified bywestern blot,protein expression level in transfection group was significantly higherthan control group.(3)The result of R302Q mutant and G100S mutant gene sequence were consistentwith expectations.2.The Scn5a over-expression vector was successfully constructed and transfected intoHEK293T cells.(1)The result of Pcdh-GFP-Scn5a sequence was consistent with the CDS sequence ofhuman Scn5a gene.(2)Forty-eight hours after transfected into HEK293T cells,green fluorescent could beseen in HEK293T cells under fluorescence microscope; total cellular RNA wasverified by real-time PCR,RNA transcript level of Scn5a in transfection group wassignificantly higher than that control group;total cellular protein was verified bywestern blot,protein expression level in transfection group was significantly higherthan control group.3.The over-expression vector Pcdh-GFP-Scn5a and The over-expression vectors p3×FLAG-CMV-7.1-PRKAG2of the wild-type PRKAG2gene, R302Q mutant and G100S mutant respectively were successfully co-transfected into HEK293T cells.(1) FLAG and GFP tag were expressed and respectively showed the red fluorescenceand green fluorescence in HEK293T cells.(2)Forty-eight hours after co-transfected into HEK293T cells, total cellular RNAtranscripts level was verified by real-time PCR, RNA transcripts of Scn5a gene weredetected in single Scn5a transfected group, Scn5a and PRKAG2gene wild-typeco-transfected group, Scn5a and R302Q mutant co-transfected group, Scn5a andG100S mutant co-transfected group,while in the control group was not detected.RNA transcripts level of PRKAG2gene was highly expressed in Scn5a and PRKAG2gene wild-type co-transfected group; while in single Scn5a transfected group,Scn5aand R302Q mutant co-transfected group, Scn5a and G100S mutant co-transfectedgroup were detected lower levels.(3)Forty-eight hours after co-transfected into HEK293T cells,total cellular protein wasverified by Western blot,Scn5a gene were expressed in single Scn5a transfected group,Scn5a and PRKAG2gene wild-type co-transfected group, Scn5a and R302Q mutantco-transfected group, Scn5a and G100S mutant co-transfected group, while in thecontrol group was not expressed.PRKAG2gene was highly expressed in Scn5a andPRKAG2gene wild-type co-transfected group; while in single Scn5a transfectedgroup,Scn5a and R302Q mutant co-transfected group, Scn5a and G100S mutantco-transfected group were expressed lower levels.ConclusionThe subject was successfully constructed PRKAG2wild-type, R302Q mutant andG100S mutant over-expression vector,meanwhile successfully constructed Scn5aover-expression vector.We successfully completed the Scn5a gene with PRKAG2gene wild-type, R302Q mutant and G100S mutant were respectively co-transfectedinto HEK293T cells.Established the foundation for further research of the function ofcardiac sodium channel affected by the mutations of PRKAG2gene.