本文選題：肌肉生長抑制素　切入點(diǎn)：CRISPR-Cas9　出處：《內(nèi)蒙古大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：肌肉生長抑制素(Myostatin, MSTN)又稱生長分化因子-8 (growth differentiation factor 8, GDF-8),是一類重要的骨骼肌細(xì)胞生長發(fā)育的負(fù)調(diào)控因子,研究表明,該基因的缺失、突變能加速肌肉生長和改變紅白肌的組成比例并增加肌肉量。CRISPR-Cas系統(tǒng)是一種來源于細(xì)菌獲得性免疫的由RNA指導(dǎo)Cas蛋白對靶向基因進(jìn)行修飾的技術(shù)。本研究利用CRISPR-Cas9系統(tǒng)對阿爾巴斯白絨山羊胎兒成纖維細(xì)胞和肌肉衛(wèi)星細(xì)胞中的MSTN基因進(jìn)行了敲除,并通過實(shí)時(shí)定量PCR與Western blot檢測了MSTN基因敲除后成纖維細(xì)胞中與之相關(guān)基因的表達(dá)情況,為通過CRISPR-Cas9系統(tǒng)生產(chǎn)基因編輯動(dòng)物奠定基礎(chǔ)。1、gRNA表達(dá)載體的設(shè)計(jì)、構(gòu)建與轉(zhuǎn)染效率分析本實(shí)驗(yàn)通過麻省理工學(xué)院的CRISPR Design (http://crispr.Mit.edu)軟件設(shè)計(jì)4對長20nt的gRNA,以gRNA-T2為模板進(jìn)行PCR,擴(kuò)增得到完整的gRNA(最終PCR產(chǎn)物長度為455bp)。為了篩選適應(yīng)于山羊細(xì)胞的最優(yōu)轉(zhuǎn)染條件,本研究利用電穿孔法將紅色熒光蛋白表達(dá)載體pCMV-DsRed導(dǎo)入絨山羊胎兒成纖維細(xì)胞中,通過流式細(xì)胞儀進(jìn)行分析的結(jié)果表明,最優(yōu)轉(zhuǎn)染條件為：每1×106個(gè)絨山羊胎兒成纖維細(xì)胞加入pCMV-DsRed質(zhì)粒10μg(1μg/μL), Opti-MEM 90μL,電壓225V,脈沖時(shí)間2.5ms。利用以上最佳轉(zhuǎn)染條件將hCas9載體和gRNA共同導(dǎo)入阿爾巴斯絨山羊胎兒成纖維細(xì)胞中,培養(yǎng)48h后提取基因組,設(shè)計(jì)跨打靶位點(diǎn)的PCR引物進(jìn)行PCR擴(kuò)增,使用Surveyor突變檢測試劑盒進(jìn)行檢測,確定有3個(gè)gRNA敲除效率較高,可以進(jìn)行下一步實(shí)驗(yàn)。2、利用CRISPR-Cas9技術(shù)制備MSTN基因敲除絨山羊胎兒成纖維細(xì)胞和肌肉衛(wèi)星細(xì)胞的的制備與分析采用最適合的轉(zhuǎn)染條件,將hCas9載體和gRNA 導(dǎo)入阿爾巴斯絨山羊胎兒成纖維細(xì)胞中和肌肉衛(wèi)星細(xì)胞中,隨機(jī)挑取單個(gè)細(xì)胞培養(yǎng),建立單克隆細(xì)胞系。對每個(gè)單克隆細(xì)胞系的基因組進(jìn)行PCR擴(kuò)增并進(jìn)行測序,篩選出靶位點(diǎn)發(fā)生突變的單克隆細(xì)胞系。本研究共獲得62個(gè)胎兒成纖維單克隆細(xì)胞系,其中有10個(gè)單等位基因敲除的單克隆細(xì)胞系和10個(gè)雙等位基因敲除的單克隆細(xì)胞系,總敲除效率為32.25%。還獲得了6個(gè)肌肉衛(wèi)星單克隆細(xì)胞系,其中有5個(gè)雙等位基因敲除的陽性單克隆細(xì)胞系,總敲除效率為83.33%。本研究選取了雙等位基因敲除的陽性成纖維細(xì)胞細(xì)胞系C205,通過qPCR對MSTN基因敲除后的肌發(fā)育相關(guān)基因表達(dá)水平進(jìn)行檢測,發(fā)現(xiàn)該細(xì)胞中MSTN基因mRNA表達(dá)量下降73.79%,Western blot的檢測結(jié)果也證實(shí)MSTN雙等位基因敲除細(xì)胞中MSTN蛋白的表達(dá)比野生型細(xì)胞MSTN蛋白的表達(dá)量減少。而隨著MSTN的表達(dá)的降低,MyoG、Myf5和Myf6等基因轉(zhuǎn)錄水平都有不同程度的上升,分別為對照細(xì)胞的1.53倍、5.68倍和3.683倍,說明MSTN對這三個(gè)基因起著負(fù)調(diào)控作用。
[Abstract]:Myostatin (MSTN), also known as growth differentiation factor-8 differentiation factor 8 (GDF-8), is an important negative regulator of skeletal muscle cell growth and development. Mutation can accelerate muscle growth, change the proportion of red and white muscle and increase muscle mass. CRISPR-Cas system is a technique of modifying target gene by Cas protein guided by RNA from bacterial acquired immunity. In this study, CRISPR-Cas9 line was used to modify the target gene. The MSTN gene was knockout from the fetal fibroblasts and muscle satellite cells of the Albus white cashmere goat. The expression of related genes in fibroblasts after MSTN gene knockout was detected by real-time quantitative PCR and Western blot, which laid a foundation for the design of gene editing vector. Construction and transfection efficiency Analysis this experiment designed 4 pairs of 20nt long gRNAs with MIT's CRISPR Design / http/ / / crispr.Mit.edu. gRNA-T2 was used as template to amplify the complete gRNAs (the final PCR product length was 455bpn. in order to screen goat cells). The optimal transfection conditions, In this study, the red fluorescent protein expression vector pCMV-DsRed was introduced into the fetal fibroblasts of cashmere goats by electroporation, and the results were analyzed by flow cytometry. The optimal transfection conditions were as follows: 1 脳 106 Cashmere Goat fetal fibroblasts were added to pCMV-DsRed plasmid 10 渭 g / 渭 L, Opti-MEM 90 渭 L, voltage 225 V, pulse time 2.5ms.Using the above optimal transfection conditions, the hCas9 vector and gRNA were co-transfected into the fetal fibroblasts of Cashmere Goat Cashmere Goat. After 48 hours of culture, genomic DNA was extracted and PCR amplification was carried out by designing PCR primers across target sites. Surveyor mutation detection kit was used to detect three gRNA knockout efficiency. The preparation and analysis of MSTN gene knockout goat fetal fibroblasts and muscle satellite cells using CRISPR-Cas9 technique can be carried out in the next step. The most suitable transfection conditions are adopted. The hCas9 vector and gRNA were introduced into the fetal fibroblasts and muscle satellite cells of the Albanian Cashmere Goat. A single cell line was selected at random and a monoclonal cell line was established. The genome of each monoclonal cell line was amplified by PCR and sequenced. In this study, 62 fetal fibroblast monoclonal cell lines were obtained, including 10 single allelic knockout and 10 double allelic knockout monoclonal cell lines. The total knockout efficiency was 32.25.The six muscle satellite monoclonal cell lines were also obtained, of which five were positive for double-allelic knockout. The total knockout efficiency was 83.33. In this study, a double allelic knockout positive fibroblast cell line, C205, was used to detect the expression level of muscle development-related genes after MSTN knockout by qPCR. It was found that the expression of MSTN gene mRNA decreased 73.79% and 73.79%. The results also confirmed that the expression of MSTN protein in MSTN double allelic knockout cells was lower than that in wild-type cells, but with the decrease of MSTN expression, the expression of MSTN protein in MSTN double allelic knockout cells was lower than that in wild-type cells. The transcriptional level of Myf6 and other genes increased in varying degrees. 1.53 times and 3.683 times of the control cells, respectively, indicating that MSTN plays a negative role in the regulation of these three genes.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S827;Q78

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相關(guān)期刊論文 前1條

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本文編號：1559801