| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 84 === Retroviral gene therapy is a technique that allows tansfer
of genes into a broad host range with high efficiency. The risk
of unpredicatable insertional mutagenesis is the major concern
in all gene transfer with random integration. To minimize the
uncertainty, we constructed a retroviral vector with positive
and negative selection modules. The enzyme cytosine deaminase
(CD), absent in mammals, catalyzes the deamination of cytosine
to uracil. It can also convert the innocuous compound
5-fluorocytosine to a highly toxic compound 5-fluorouracil,
which has been proved lethal to the cells and is widely used in
cancer chemotherapy. The cells expressing the bacterial gene for
cytosine deaminase would be eliminated in the presence of
5-fluorocytosine. Combined with neomycin phosphotransferase
(neo) as the positive selection module, a retroviral vector
containing neo and CD genes has been constructed. The cells
infected by this vector show higher susceptibility to
5-fluorocytosine compared with those infected by their control
counterpart. The lower tumor incidence has also be observed in
the animal experiments which simulated the unpleasant occurrence
of tumor after gene transfer. Furthermore, to provide more space
for accommodation of foreign cDNA, a retroviral vector
accompanied with a novel positive selection scheme for the
existence of bacterial CD has been established. The CD gene
could serve not only as positive but also as negative selection
modules for cells transfected by retroviral vector containing CD
gene itself. This strategy might facilitate the possibility to
control the cells with retroviral infections after furnishing
their effect of the transgene either in vivo or ex vivo.
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