Expression of hepatitis B virus X protein in transgenic mice.
OBJECTIVE
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-HBx was 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-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene 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.


CONCLUSIONS
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 HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.
Xiong
Jun
J
Department of Cell Biology, Second Military Medical University, Shanghai 200433, China.
Yao
Yu-Cheng
YC
Zi
Xiao-Yuan
XY
Li
Jian-Xiu
JX
Wang
Xin-Min
XM
Ye
Xu-Ting
XT
Zhao
Shu-Min
SM
Yan
Yong-Bi
YB
Yu
Hong-Yu
HY
Hu
Yi-Ping
YP
D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006509:Hepatitis B / Q000235:genetics; D006511:Hepatitis B Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000648:ultrastructure / Q000821:virology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D006511:Hepatitis B Antigens; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein