[The construction of recombinant AAV vector expressing HSVtk gene controlled by Tet-On and the detection of its activity].
In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Chen
Qian
Q
Molecular Biology Research Center, College of Biological Science and Technology, Central South University, Changsha 410078, China.
Li
Zi-Bo
ZB
Zeng
Zhao-Jun
ZJ
Luo
Sai-Qun
SQ
Hu
Wei-Xin
WX
D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D013937:Thymidine Kinase; D004318:Doxycycline; D015774:Ganciclovir