Establishment of an effectively lac operator- repressor based inducible RNAi system in mammalian cells
| Autor: | Ren-Huang Wu, 吳仁煌 |
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| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 RNA interference (RNAi) is mediated by small interfering RNAs that target and degrade mRNA in a sequence-specific manner. However, the down-regulation of essential genes by RNAi will result in an arrest of cell growth or in cell death, thus imposing significant limitations on the applications of RNAi to long-term studies on the function of these genes. Availability of an inducible RNAi system will overcome this limitation. The controlled expression of short hairpin RNAs (shRNAs) molecules in a temporal and spatial manner would be bentifical for studying loss-of-function phenotypes in the context of cellular development and differentiation. Here, we describe an inducible small interfering RNAs (siRNAs) expression system that allows a significant control of the specific gene silencing by RNAi. First, we generated several constructs composed of the human H1 promoter and operator sequence derived from the Escherichia coli lac operator-repressor system. In this inducible RNAi system, lac repressor would bind to operator in the absence of isopropyl-β-D-1- thiogalactoside (IPTG) to repress gene expression and lac operator in these constructs was varied to determine the most effective combination for optimal repression/induction. In addition, we used the firefly luciferase reporter gene and p53 gene as the target to determine the efficiency of our inducible RNAi system. The activity of luciferase reporter was reduced by 40% in the absence of IPTG and reduced by 95% after IPTG treatment. Therefore, the lac repressor could bind to operator sequence and inhibited the expression of shRNAs transcripted by modified H1 promoter. Although these constructs could mediate lac repressor and IPTG regulation to a certain extent, significant levels of background expression of shRNA were observed in these constructs. To overcome the observed leaky expression from these constructs, we generated tandem-type siRNA expression constructs composed of the modified H1 promoter. These constructs showed the features of tight repression of transcription in the absence of IPTG and the rapid induction of transcription on addition of IPTG. Using this system, we demonstrated the inducible RNAi effect on the gene expression in Baby Hamster Kidney cell (BHK-21). In summary, our inducible RNAi system should be useful both for basic researches on gene function and therapeutic applications. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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