| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 89 === Retroviral transduction is a technique for introducing genes of interest into mammalian cells. The genes introduced this way can integrate into host chromosome and express stably. Moreover, retrovirus can transduce primary cells that are difficult to receive genes delivered by conventional methods. My project has aimed to establish retroviral transduction of murine primary lymphocytes. The first part of my work was to produce high titer recombinant retrovirus. PhoenixE cell line carrying plasmids encoding retroviral core(gag)/polymerase(pol) or envelope(env) genes was used to produce recombinant virus after transfecting viral vector which contains retroviral packaging signal(y) and the gene of insterest. Two viral vectors, pBMN-EGFP and pGC-YFP, were used to transfect the packaging cell line. I determined the optimal amounts of plasmid DNA and chloroquine for CaPO4 transfection. Then, I tested conditions for spin infection. Finally, I could produce the recombinant retrovirus with a titer around 1x106 unit/ml.
The second part of my work was using the retrovirus to transduce murine lymphocytes. Three types of murine lymphocytes were used. The first cell type was total spleen cells activated by PMA&ionophore. Twenty-two percent of activated spleenocytes were transduced as determined by YFP expression. The second cell type was TCRgd+ intestinal intraepithelial lymphocytes (gdiIEL) activated by anti-TCRgd Ab. Ten percent of gd-iIEL expressing YFP during primary activation were transduced. The third cell type was spleen and lymph node CD8 cells activated with immobilized anti-TCRb Ab&anti-CD28 Ab. After retrovirus transduction, fifteen percent of activated CD8 cells expressed YFP.
The established experimental conditions will allow us to introduce gene of interest into gdiIEL and CD8 cells to study gene function.
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