Biochemical and Biophysical Research Communications
Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors
Section snippets
Materials and methods
Construction of adenovirus. All adenoviruses used were created in 293 cells by using pHVad2 [8] as the left-end shuttle plasmid with pBHG10 (Microbix, Toronto, Canada) as the right end. Briefly, the SV40 polyadenylation signal was removed from pRep4 (Invitrogen, Carlsbad, CA) by XhoI and SalI digestion and cloned into the SalI site pHVad2, generating pHV.SV40pA. We created pHV.AP.SV40pA by removing the AP gene from pLAPSN [9] by digestion with EcoRI and Bpu10I, blunting, and inserting it into
Results and discussion
Transcriptional targeting is a means to restrict gene expression from Ad vectors to a particular cell type [15]. In our studies, we used a modified version of the AFP promoter to achieve transgene expression specifically in HCC cells. The AFP promoter contains two enhancer elements, which have been previously demonstrated to be activated specifically in AFP-expressing HCC cell lines [16]. The promoter also includes six copies of a silencer element, responsible for silencing the promoter in
Acknowledgements
Xun Ye and Min Liang contributed equally to this work. We thank Kathrin Bernt for help with the in vitro transduction studies; A. Taketa and J. Sato for the modified version of the AFP promoter. This work was supported in part by national 863 Grant 2001AA217131 (LM) and NIH Grant R01 CA 80192 (AL).
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