本文關(guān)鍵詞： 肌肉生成抑制素 shRNA 可控表達(dá) 轉(zhuǎn)基因小鼠　出處：《揚(yáng)州大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：MSTN是肌肉生長(zhǎng)發(fā)育的一個(gè)重要的負(fù)調(diào)控因子,是影響體型發(fā)育的經(jīng)典基因之一,其表達(dá)量的增加與肌肉量的減少密切相關(guān),抑制MSTN基因的功能,動(dòng)物會(huì)呈現(xiàn)出雙肌表型,這對(duì)畜牧生產(chǎn)、動(dòng)物遺傳育種、肌肉萎縮性疾病的診斷、治療和預(yù)后等方面具有重要意義,但MSTN是一個(gè)在胚胎早期就能表達(dá)的基因,胚胎期沉默或敲除MSTN基因會(huì)因肌肉過度肥大而造成難產(chǎn)、流產(chǎn)、死胎。實(shí)現(xiàn)MSTN基因的可控表達(dá)成為發(fā)揮MSTN基因應(yīng)用價(jià)值的關(guān)鍵。本研究通過設(shè)計(jì)MSTN shRNA并驗(yàn)證其基因沉默效率,構(gòu)建可控表達(dá)MSTN shRNA載體,制備高效可誘導(dǎo)MSTN shRNA表達(dá)的轉(zhuǎn)基因小鼠模型,利用可控表達(dá)轉(zhuǎn)基因小鼠模型研究MSTN對(duì)肌肉生長(zhǎng)發(fā)育的影響,為將可控表達(dá)的MSTN基因應(yīng)用到基因工程育種、藥物治療肌肉萎縮病等方面奠定技術(shù)基礎(chǔ),提供有價(jià)值的參考。主要研究?jī)?nèi)容和結(jié)果如下：研究一：小鼠MSTN shRNA過表達(dá)載體構(gòu)建及基因沉默效率檢測(cè)本研究根據(jù)小鼠MSTN mRNA序列設(shè)計(jì)了4對(duì)shRNA序列,構(gòu)建相應(yīng)的MSTN shRNA過表達(dá)載體pGPU6/GFP/Neo-MSTN shRNA,利用QRT-PCR、Western blot技術(shù)檢測(cè)shRNA干擾后細(xì)胞內(nèi)MSTN mRNA及蛋白表達(dá)水平。結(jié)果表明,本研究設(shè)計(jì)并構(gòu)建的4個(gè)MSTN shRNA過表達(dá)載體,在細(xì)胞水平都能有效降低MSTN mRNA及蛋白的表達(dá)水平,其中轉(zhuǎn)染pGPU6/GFP/Neo-MSTN714載體細(xì)胞組基因沉默效率最高,MSTN mRNA和蛋白表達(dá)分別下降72%、74%,差異極顯著(P0.01)。研究二：小鼠MSTN shRNA可控表達(dá)載體構(gòu)建及誘導(dǎo)效率檢測(cè)選取干擾效率最好的shRNA MSTN714構(gòu)建可控表達(dá)載體ptTS-MSTN shRNA,重組載體轉(zhuǎn)染小鼠成肌細(xì)胞經(jīng)G418篩選富集整合有外源載體的細(xì)胞,通過強(qiáng)力霉素誘導(dǎo)細(xì)胞中MSTN shRNA表達(dá),利用細(xì)胞免疫熒光觀察細(xì)胞內(nèi)綠色熒光蛋白,利用QRT-PCR、 Western blot技術(shù)檢測(cè)MSTN mRNA及蛋白的表達(dá)水平檢測(cè)誘導(dǎo)效率。結(jié)果表明,tTS-MSTN shRNA組細(xì)胞和Control組細(xì)胞在不添加DOX時(shí),僅檢測(cè)到少量綠色熒光表達(dá),添加DOX后,GFP蛋白信號(hào)顯著增強(qiáng)；經(jīng)DOX誘導(dǎo)后,轉(zhuǎn)染含MSTN shRNA重組載體細(xì)胞組MSTN mRNA相對(duì)表達(dá)水平比未經(jīng)DOX誘導(dǎo)組及其他對(duì)照組細(xì)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(dá)量差異不顯著(P0.05)。研究三：可控表達(dá)MSTN shRNA轉(zhuǎn)基因小鼠的制備及對(duì)小鼠生長(zhǎng)發(fā)育的評(píng)價(jià)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(dá)轉(zhuǎn)基因小鼠。利用PCR技術(shù)對(duì)獲得的FO代轉(zhuǎn)基因小鼠進(jìn)行整合檢測(cè),利用QRT-PCR方法檢測(cè)轉(zhuǎn)基因小鼠體內(nèi)外源基因tTS的表達(dá)。檢測(cè)結(jié)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(dá)效率最好的轉(zhuǎn)基因小鼠擴(kuò)繁的后代進(jìn)行轉(zhuǎn)基因小鼠MSTN shRNA誘導(dǎo)活性、生長(zhǎng)狀況、繁殖性能、肌肉組織學(xué)等進(jìn)行檢測(cè)評(píng)價(jià)。結(jié)果表明,經(jīng)DOX誘導(dǎo)的Tg+DOX組小鼠體重顯著低于Tg、WT+DOX、WT組小鼠；所制備的轉(zhuǎn)基因小鼠,DOX誘導(dǎo)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(dǎo)的MSTN轉(zhuǎn)基因小鼠及正常小鼠相比,加DOX誘導(dǎo)之后的MSTN轉(zhuǎn)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(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.2

【參考文獻(xiàn)】

相關(guān)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(duì)鵝PRL和PRLR基因表達(dá)的影響[J];揚(yáng)州大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版);2014年04期

2 鞠輝明;白立景;姜星;李奎;;整合型誘導(dǎo)表達(dá)豬生長(zhǎng)激素(pGH)載體的構(gòu)建及表達(dá)研究[J];畜牧獸醫(yī)學(xué)報(bào);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(gòu)建及干涉效率的檢測(cè)[J];黑龍江畜牧獸醫(yī);2012年07期

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本文編號(hào)：1534002