Expression of hepatitis B virus X protein in transgenic mice

AIM: To establish a mice model harboring hepatitis B virusx gene (adr subtype) for studying the function of hepatitis Bvirus X protein, a transactivator of viral and cellular promoter/enhancer elements.METHODS: Expression vector pcDNA3-HBx, containing CMVpromoter and hepatitis B virus x gene open reading fragment,was constructed by recombination DNA technique. Hela cellswere cultured in DMEM and transfected with pcDNA3-HBxor control pcDNA3 plasmids using FuGENE6 TransfectionReagent. Expression of pcDNA3-HBx vectors in thetransfected Hela cells was confirmed by Western blotting.After restriction endonudease digestion, the coding elementswere microinjected into male pronuclei of mice zygotes. Thepups were evaluated by multiplex polymerase chain reaction(PCR) at genomic DNA level. The x gene transgenic micefounders were confirmed at protein level by Western blotting,immunohistochemistry and immunogold transmissionelectron microscopy.RESULTS: Expression vector pcDNA3-HBxwas constructedby recombination DNA technique and identified right byrestriction endonuclease digestion and DNA directsequencing. With Western blotting, hepatitis X protein wasdetected in Hela cells transfected with pcDNA3-HBxplasmids,suggesting pcDNA3-HBxplasmids could express in eukaryoticcells. Following microinjection of coding sequence ofpcDNA3-HBx, the embryos were transferred to oviducts ofpsedopregnant females. Four pups were born and survived.Two of them were verified to have the HBxgene integratedin their genomic DNA by multiplex PCR assay, and namedC57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3respectively. They expressed 17KD X protein in liver tissueby Western blotting assay. With the immunohistochemistry,X protein was detected mainly in hepatocytes cytoplasm oftransgenic mice, which was furthermore confirmed byimmunogold transmission electon microscopy.CONCLUSION: We have constructed the expression vectorpcDNA3-HBxthat can be used to study the function of HBxgene in eukaryotic cellsin vitro. We also established HBxgene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBxgene in their genome and express Xprotein in hepatocytes, Which might be a valuable animalsystem for studying the roles of HBxgene in hepatitis B viruslife cycle and development of hepatocellular carcinoma in vivo.

AIM:To establish a mice model harboring hepatitis B virus x gene(adr subtype)for studying the function of hepatitis B virus X protein,a transactivator of viral and cellular promoter/ enhancer elements. METHODS:Expression vector pcDNA3-HBx,containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique.Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent.Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion,the coding elements were microinjected into male pronuclei of mice zygotes.The pups were evaluated by multiplex polymerase chain reaction (PCR)at genomic DNA level.The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS:Expression vector pcDNA3-HBxwas constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing.With Western blotting,hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBxplasmids could express in eukaryotic cells.Following microinjection of coding sequence of pcDNA3-HBx,the embryos were transferred to oviducts of psedopregnant females.Four pups were born and survived. Two of them were verified to have the HBxgene integrated in their genomic DNA by multiplex PCR assay,and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively.They expressed 17KD X protein in liver tissue by Western blotting assay.With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice,which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION:We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro.We also established HBx gene(adr subtype)transgenic mice named C57-TgN(HBx) SMMU harboring HBxgene in their genome and express X protein in hepatocytes,Which might be a valuable animal system for studying the roles of HBxgene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.