[Adeno-associated virus vector mediated gene transfer of HSVI-TK and its effect on killing cancer cell].
The plasmid pACTK-19 was constructed by inserting HSVI-TK gene into the multiple cloning sites of the general adeno-associated virus(AAV) vector pACR-Neo. When plasmid pACTK-19 was transfected to recombinant AAV's packaging cell line AE1201, which was exposed to Adenovirus 5 for two hours before transfection, we got rAAV/ACTK at the titer of 3.4 x 10(5) CFU/ml. After infecting human lung cancer cell A549 with rAAV/ACTK, we extracted host cell's chromosome cDNA and amplified part of the HSVI-TK sequence by a pair of HSVI-TK's primers and then hybridized to digoxin labelled HSVI-TK gene probe. We got corresponding positive band and it proved the integration of HSVI-TK into host cell's chromosome cDNA. By reverse transcripting rAAV/ACR-Neo infected A549 total cell RNA, we amplified part of HSVI-TK sequence and hybridized to Neo gene probe. The corresponding positive band demonstrated the expression of HSVI-TK. So through rAAV/ACTK, HSVI-TK was transferred into host cells and was expressed in them, the rAAV's genome was integrated into host cell's chromosome DNA. Connecting with Ganciclovir (GCV), the HSVI-TK gene transfer mediated by rAAV/ACTK, could inhibit the synthesis of chromosome DNA and lead to the killing of the lung cancer cell. This work proved that recombinant AAV could be used as vector for gene transduction to cancer cell, and also laid foundation for further research and application of AAV vector.
Yao
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E
National Lab of Molecular Virology and Genetic Engineering, Beijing 100052.
Feng
Z
Z
Wu
X B
XB
D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D015774:Ganciclovir / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D013937:Thymidine Kinase; D015774:Ganciclovir