碩士 / 國立臺灣大學 / 分子與細胞生物學研究所 / 96 / RNA interference (RNAi) is a powerful technique to silence target gene in vitro. Short hairpin RNA (shRNA) is driven by RNA polymerase Ⅲ, resulting that it does not enable to be transcribed in a specific tissue to inhibit gene. Overexpression of shRNA causes death of the organism. Therefore, a system that allows short interfering RNA (siRNA) be regulated by RNA polymerase II is required. We take advantage of using IE1 (+502/+527) and IE2 (+816/+835) of zebrafish myf5 because we proved that the DNA fragment with stem-loop structure that ranged from 76 bp to 97 kb between IE1 and IE2 were cut out in RNA level. We constructed a plasmid pβ-actin::DsRed-IE1-GFP shRNA-IE2, in which reporter RFP is driven by β-actin promoter and shRNA corresponding to GFP mRNA(+416/+437) is inserted between IE1 and IE2, and co-transfected with another plasmid, pCMV::GFP, in which GFP reporter gene is driven by the enhancer and promoter of cytomegarovirus, into COS-1 cells. Results showed that GFP expressing cell number was suppressed to 40 %, compared to cells that were co-transfected with pβ-actin::DsRed and pCMV::GFP. In addition, two plasmids were constructed: plasmid pcMLC2::GFP-IE1-pre-let7-IE2, in which pre-let7 from zebrafish is inserted into two IE motifs, fused with GFP and driven by a heart-specific promoter cmlc2, and plasmid pcMLC2::luc-lin41-3’UTR, in which 3’UTR of lin41 is fused with luciferase and driven by cmlc2 promoter. After co-injection of these two plasmids into zebrafish embryos, we found that the luciferase activity was decreased to 10 % of that was displayed in the embryos co-injected with pcMLC2::GFP-IE1-pre-let7-IE2 and pcMLC2::luc. Furthermore, we constructed a plasmid pVMHC::IE1- cTnnt2 shRNA–IE2, in which shRNA that targets specifically to zebrafish cardiac troponin T2 (cTnnt2) is driven by ventricle-specific promoter, ventricular myosin heavy chain (VMHC), and microinjected into zebrafish embryos. We found that the pVMHC::IE1-cTnnt2 shRNA–IE2-injected embryos appeared the ventricle defects, such as weak contractile and strung heart, which mimicked the phenotypes induced by injection of cTnnt2-morpholino. We then constructed a plasmid pCMLTet-IE1-cTnnt2 shRNA-IE2, in which IE1-cTnnt2 shRNA-IE2 was constructed in a heart-specifically expressed Tet-on system. By linearizing the plasmid and injected it into one-cell stage zebrafish embryo, we generated transgenic lines that carried pCMLTet-IE1-cTnnt2 shRNA-IE2. After doxycycline induction, F1 transgenic zebrafish embryos could expressed GFP reporter gene and IE1-cTnnt2 shRNA-IE2 specifically in heart, and we found that cTnnt2 mRNA and cTnnt2 protein expressed in heart were dramatically suppressed. Taken together, we conclude that IE1 and IE2 enable to serve as two cassettes to carry a shRNA, and this shRNA enables to be released and to silence target gene under the control of tissue-specific and regulable RNA polymerase II. This system might be highly potential to be applied in gene therapy.
| Identifer | oai:union.ndltd.org:TW/096NTU05061013 |
| Date | January 2008 |
| Creators | Chieh-Lin Wu, 吳杰霖 |
| Contributors | Huai-Jen Tsai, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 68 |
Page generated in 0.0072 seconds