蔡懷楨Tsai, Huai-Jen臺灣大學:分子與細胞生物學研究所吳杰霖Wu, Chieh-LinChieh-LinWu2010-06-022018-07-062010-06-022018-07-062008U0001-2207200811271800http://ntur.lib.ntu.edu.tw//handle/246246/184689RNA interference (RNAi)是一項能夠在in vitro環境下有效抑制目標基因表現的技術。直接將合成的short interfering RNA (siRNA)轉染到細胞中，或是利用RNA polymerase Ⅲ promoter在細胞內驅動short hairpin RNA (shRNA)表現，均可有效地抑制目標基因表現。然而，現行報告中利用RNA polymerase Ⅲ驅動shRNA表現的RNAi系統，這些不具有組織專一性且無法進行誘導的系統，通常會導致過度表現shRNA而造成生物體死亡。所以，建構由RNA polymerase II驅動的可調控RNAi系統是必須的。於是我們利用斑馬魚myf5基因intronic RNA的IE1(+502/+527)和IE2(+816/+835)，因其之間帶有具二級結構的約76 bp到97 bp大小的DNA片段，經由RNA polymerase Ⅱ驅動後能在RNA層級下被剪輯出來，並在活體中表現 shRNA進行RNAi的功能。首先構築RFP (DsRed)報導基因下游接著IE motifs與能抑制GFP mRNA表現的GFP shRNA，並藉由β-actin promoter驅動的pβ-actin::DsRed-IE1-GFP shRNA-IE2質體；並藉由CMV promoter驅動GFP報導基因表現的pCMV::GFP質體共同轉染COS-1 cells。結果發現相較於共同轉染pβ-actin::DsRed與pCMV::GFP的控制組，綠色螢光表現的細胞數目下降了60%。進一步地，我們構築了由心臟專一性cMLC2 promoter驅動GFP報導基因，下游接著攜帶斑馬魚pre-microRNA let-7 (pre-let7)的IE1和IE2 motif之質體，pcMLC2:: GFP-IE1-pre-let7-IE2；與另外一個由cMLC2 promoter驅動下游接有luciferase (luc)以及let7結合序列lin41 3’UTR的質體，pcMLC2::luc-lin41 3’UTR。將這兩個質體共同注射到斑馬魚胚胎並偵測luciferase活性，結果發現相較控制組，共同注射pcMLC2::GFP-IE1 -pre-let7-IE2與pcMLC2::luc的luciferase活性下降了90%。同樣地，我們又構築能抑制斑馬魚cardiac troponin T2 (cTnnt2)的shRNA接在IE1與IE2之間，並由心室專一性的ventricular myosin heavy chain (VMHC) promoter驅動表現，得到pVMHC::IE1-cTnnt2 shRNA–IE2質體。將質體注射到斑馬魚胚胎後發現，心室在發育過程中出現收縮強度變弱、心臟looping不完全等缺陷情形。這與利用胚胎發育抑制劑 (morpholino) knockdown cTnnt2表現的胚胎有類似的表型。最後我們將IE1-cTnnt2 shRNA-IE2構築到能在心臟專一性表現的Tet-on系統而得到pCMLTet-IE1-cTnnt2 shRNA-IE2質體，並挑選穩定表現的基因轉殖斑馬魚。在8 hpf至7 dpf以doxycycline誘導，發現帶有轉殖基因的F1胚胎能在心臟同時表達出綠螢光報導基因與IE1-cTnnt2 shRNA-IE2，並且造成胚胎心臟中cTnnt2 mRNA與cTnnt2蛋白質表現量都顯著地受到抑制。從上述研究我們認為，IE1與IE2確實可以被用來作為攜帶shRNA的cassette，再利用具組織專一性與可調控性的RNA polymerase Ⅱ promoter來驅動，如此就可在特定組織中、特定時期將特定基因表現knockdown。這套可調控性表現RNAi的系統，或許可以將來有潛力地應用在基因治療上。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.中文摘要 ----------------------------------------------- 1文摘要 ----------------------------------------------- 4言 --------------------------------------------------- 6驗材料與方法 ----------------------------------------- 12果 --------------------------------------------------- 26論 --------------------------------------------------- 36考文獻 ----------------------------------------------- 46表 --------------------------------------------------- 51錄 --------------------------------------------------- 63application/pdf1861520 bytesapplication/pdfen-US斑馬魚RNA干擾shRNARNAiZebrafishhttp://ntur.lib.ntu.edu.tw/bitstream/246246/184689/1/ntu-97-R95b43028-1.pdf