本文關鍵詞： 肌肉生成抑制素 shRNA 可控表達 轉基因小鼠　出處：《揚州大學》2016年碩士論文　論文類型：學位論文

【摘要】：MSTN是肌肉生長發(fā)育的一個重要的負調(diào)控因子,是影響體型發(fā)育的經(jīng)典基因之一,其表達量的增加與肌肉量的減少密切相關,抑制MSTN基因的功能,動物會呈現(xiàn)出雙肌表型,這對畜牧生產(chǎn)、動物遺傳育種、肌肉萎縮性疾病的診斷、治療和預后等方面具有重要意義,但MSTN是一個在胚胎早期就能表達的基因,胚胎期沉默或敲除MSTN基因會因肌肉過度肥大而造成難產(chǎn)、流產(chǎn)、死胎。實現(xiàn)MSTN基因的可控表達成為發(fā)揮MSTN基因應用價值的關鍵。本研究通過設計MSTN shRNA并驗證其基因沉默效率,構建可控表達MSTN shRNA載體,制備高效可誘導MSTN shRNA表達的轉基因小鼠模型,利用可控表達轉基因小鼠模型研究MSTN對肌肉生長發(fā)育的影響,為將可控表達的MSTN基因應用到基因工程育種、藥物治療肌肉萎縮病等方面奠定技術基礎,提供有價值的參考。主要研究內(nèi)容和結果如下：研究一：小鼠MSTN shRNA過表達載體構建及基因沉默效率檢測本研究根據(jù)小鼠MSTN mRNA序列設計了4對shRNA序列,構建相應的MSTN shRNA過表達載體pGPU6/GFP/Neo-MSTN shRNA,利用QRT-PCR、Western blot技術檢測shRNA干擾后細胞內(nèi)MSTN mRNA及蛋白表達水平。結果表明,本研究設計并構建的4個MSTN shRNA過表達載體,在細胞水平都能有效降低MSTN mRNA及蛋白的表達水平,其中轉染pGPU6/GFP/Neo-MSTN714載體細胞組基因沉默效率最高,MSTN mRNA和蛋白表達分別下降72%、74%,差異極顯著(P0.01)。研究二：小鼠MSTN shRNA可控表達載體構建及誘導效率檢測選取干擾效率最好的shRNA MSTN714構建可控表達載體ptTS-MSTN shRNA,重組載體轉染小鼠成肌細胞經(jīng)G418篩選富集整合有外源載體的細胞,通過強力霉素誘導細胞中MSTN shRNA表達,利用細胞免疫熒光觀察細胞內(nèi)綠色熒光蛋白,利用QRT-PCR、 Western blot技術檢測MSTN mRNA及蛋白的表達水平檢測誘導效率。結果表明,tTS-MSTN shRNA組細胞和Control組細胞在不添加DOX時,僅檢測到少量綠色熒光表達,添加DOX后,GFP蛋白信號顯著增強；經(jīng)DOX誘導后,轉染含MSTN shRNA重組載體細胞組MSTN mRNA相對表達水平比未經(jīng)DOX誘導組及其他對照組細胞中降低70%左右,差異極顯著(P0.01),MSTN蛋白也下降90%左右,其他各組間MSTN mRNA及蛋白表達量差異不顯著(P0.05)。研究三：可控表達MSTN shRNA轉基因小鼠的制備及對小鼠生長發(fā)育的評價將tTS-MSTN shRNA載體線性化后,利用顯微注射法制備MSTN shRNA可控表達轉基因小鼠。利用PCR技術對獲得的FO代轉基因小鼠進行整合檢測,利用QRT-PCR方法檢測轉基因小鼠體內(nèi)外源基因tTS的表達。檢測結果表明,制備的159只F0代小鼠中得到8只陽性小鼠。選取tTS表達效率最好的轉基因小鼠擴繁的后代進行轉基因小鼠MSTN shRNA誘導活性、生長狀況、繁殖性能、肌肉組織學等進行檢測評價。結果表明,經(jīng)DOX誘導的Tg+DOX組小鼠體重顯著低于Tg、WT+DOX、WT組小鼠；所制備的轉基因小鼠,DOX誘導前的產(chǎn)仔數(shù)(7±0.637.2±0.84)和窩存活仔數(shù)(6.2±0.756.4±0.55)均與同期飼養(yǎng)的正常小鼠(WT)差異不顯著(P0.05)；與未經(jīng)誘導的MSTN轉基因小鼠及正常小鼠相比,加DOX誘導之后的MSTN轉基因小鼠肌纖維數(shù)目增多、肌纖維橫截面積增大。
[Abstract]:MSTN is an important negative regulator of muscle growth, is one of the classic genetic effect of somatotype growth, its expression increased and muscle volume reduction is closely related to the inhibition of MSTN gene function, the animal will show a double muscle phenotype, the animal husbandry, animal genetic breeding, disease diagnosis of muscle atrophy, is of great significance for treatment and prognosis, but MSTN is an early gene expression can embryo, embryo silencing or knocking out the MSTN gene dystocia, caused by excessive muscle hypertrophy, abortion, stillbirth. Controllable expression of MSTN gene MSTN gene play has become a key application value. This study by the design of MSTN shRNA and verify the gene silencing efficiency, build a controlled MSTN expression vector shRNA, transgenic mice model, can induce MSTN shRNA expression, the expression of MST in transgenic mouse model with controllable Effect of N on the growth of muscle, the controllable expression of MSTN gene to gene engineering breeding, lay the foundation treatment of muscle atrophy disease, provide a valuable reference. The main research contents and results are as follows: A: mouse MSTN shRNA expression carrier construction and gene silencing efficiency detection based on mouse MSTN mRNA sequence designed 4 pairs of shRNA sequences, constructed MSTN shRNA expression vector pGPU6/GFP/Neo-MSTN shRNA, using QRT-PCR, MSTN and mRNA protein levels in cells to detect the expression of shRNA Western interference blot technology. The results showed that 4 MSTN shRNA on the design and construction of the expression vector, can effectively reduce the expression level MSTN mRNA and protein at the cell level, which was transfected into pGPU6/GFP/Neo-MSTN714 cell carrier group gene silencing efficiency is the highest, the expression of MSTN mRNA and protein were decreased by 72 %, 74%, significant difference (P0.01). Study two: mouse MSTN shRNA controlled expression vector construction and induction efficiency of detection of selected jamming efficiency best shRNA MSTN714 to build the controllable expression vector ptTS-MSTN shRNA. The recombinant vector was transfected into mouse myoblasts by G418 screening of exogenous vector cells enriched by integration, doxycycline induced MSTN expression in shRNA cells in the use of green fluorescent protein was observed by immunofluorescence in cells, using QRT-PCR to detect the expression of Western blot detection of MSTN mRNA and protein induction efficiency. The results showed that tTS-MSTN cells in shRNA group and Control group of cells without DOX, only detected the expression of green fluorescence, the addition of DOX, GFP protein signal significantly enhanced; after induced by DOX, MSTN transfected with shRNA recombinant vector MSTN mRNA cell group relative expression levels than those induced by DOX group and other cells in the control group In the reduced about 70%, significant difference (P0.01), MSTN protein was also decreased by about 90%, other groups of MSTN mRNA and protein expression were not significant (P0.05). Study three: preparation of MSTN controlled expression of shRNA transgenic mice and to evaluate the growth and development of mouse tTS-MSTN shRNA vector linearized by MSTN shRNA controlled expression of transgenic mice by microinjection method. The integration of detection of FO generation of transgenic mice by using PCR technology, using QRT-PCR method to detect the expression of exogenous tTS gene in transgenic mice in vivo. The detection results show that the obtained 8 transgenic mice produced 159 F0 mice. The expression of tTS in transgenic mice was selected the best the efficiency of the offspring of transgenic mice MSTN induced shRNA activity, growth status, reproductive performance, muscle histology were examined. The results showed that the Tg+ DOX mice induced by DOX. Weight was significantly lower than that of Tg, WT+DOX, WT group of mice; transgenic mice were prepared, DOX before the induction of the litter number (7 + 0.637.2 + 0.84) and nest survival litter (6.2 + 0.756.4 + 0.55) were compared with normal mice fed (WT) had no significant difference (P0.05); and without MSTN transgenic mice and normal mice induced by MSTN transgenic mice compared to the number of muscle fibers after DOX induced increase in muscle fiber cross-sectional area increased.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S852.2

【參考文獻】

相關期刊論文 前3條

1 王健;張宜輝;張蕊;龔道清;王利剛;陳宏軍;;反季節(jié)調(diào)控對鵝PRL和PRLR基因表達的影響[J];揚州大學學報(農(nóng)業(yè)與生命科學版);2014年04期

2 鞠輝明;白立景;姜星;李奎;;整合型誘導表達豬生長激素(pGH)載體的構建及表達研究[J];畜牧獸醫(yī)學報;2013年07期

3 安星蘭;薛超;樸善花;苗向陽;滕春波;安鐵洙;王春生;;小鼠MSTN基因RNA干涉載體的構建及干涉效率的檢測[J];黑龍江畜牧獸醫(yī);2012年07期

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