A novel strategy of establishing an in vitro assay systemin DF-1 and NIH3T3 cell lines
| Autor: | Ming-Hua Mao, 毛敏驊 |
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| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 To react to extracellular stimuli, cells employ various mechanisms to regulate different cellular responses, which usually require synthesis of novel mRNAs and proteins. Real-time PCR has provided valuable quantitative information on the regulation of cellular responses at mRNA level; however those results are not applicable to proteins that have translational or post-translational control. To obtain accurate and reproducible quantitative information on how cells respond to extracellular signals at protein level, we found the conventional transient transfection strategy that can not precisely measure transcription factors activity is not sufficient. Therefore, we intend to develop a platform that combines Cre/loxP system and dual luciferase reporter system for quantitative analysis of transcription factors activity in vitro and in vivo. To minimize cellular variations, we generated stable cell lines for firefly luciferase reporters, and used Renilla luciferase-fused transcription factor to measure the quantity of individual transcription factor. Also to facilitate the time-consuming processes of generating multiple stable cell lines, we used Cre/loxP system for efficient target sequence swapping. To demonstrate the practical applications of this strategy, we generated a collection of reporter cell lines from DF-1 and NIH/3T3 cells containing lox sites for rapid exchange of target DNA sequences. Further, this system can be applied to study the function of different proteins in transgenic animals. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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