| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 99 === Apoptosis, the programed cell death, plays an critical role in cancer therapy currently. BikDD, a T33D/ S35D mutant of pro-apoptotic protein Bik, has been used to eliminate cancer cells in pancreatic and lung cancers. Therefore, we hope to expand the application of BikDD to not only in cancer therapy but also in the researches of embryonic development and regenerative medicine. Fisrt, we designed a temporally and spatially controlled tissue-specific cell ablation system, combining BikDD and inducible tetracycline-controlled transcriptional activation (tet-on) system, named tet-on-BikDD cell ablation system. Then, a zebrafish transgenic line ZCtBIBCM, which possessed tet-on-BikDD cell ablation system, was established. In ZCtBIBCM embryos, rtTA-M2 was driven by zebrafish cardiac myosin light chain2 (cmlc2) promoter, and after rtTA-M2 combined with doxycycline (Dox), the active rtTA-M2 activated tetO min CMV promoter droved BikDD in cardiomyocytes specifically. After Dox treated, tubular and collapsed hearts showed, and the GFP positive cardiomyocytes was decreased 70 % in ZCtBIBCM embryos. Furthermore, In whole mount in-situ hybridization (WISH), BikDD was detected in cardiomyocytes specifically. And the reduction of cardiomyocytes resulted from cell apoptosis induced by BikDD was proved in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, we have observed 51.2 % (43 / 84 ) of embryos with injured hearts can recover after Dox removed, that meant tet-on-BikDD cell ablation system was reversible. These evidences indicated that the tet-on-BikDD cell ablation system is effective for tissue-specifically, temporally and spatially controlled cell ablation in zebrafish, thereby might be a potential genetic tool to analyze tissue interactions in embryonic development as well as the mechanisms of regeneration.
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