發(fā)布時間：2018-03-03 06:00

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

【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胞生長發(fā)育的負調(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)是一種來源于細菌獲得性免疫的由RNA指導Cas蛋白對靶向基因進行修飾的技術(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胞和肌肉衛(wèi)星細胞中的MSTN基因進行了敲除,并通過實時定量PCR與Western blot檢測了MSTN基因敲除后成纖維細胞中與之相關(guān)基因的表達情況,為通過CRISPR-Cas9系統(tǒng)生產(chǎn)基因編輯動物奠定基礎(chǔ)。1、gRNA表達載體的設(shè)計、構(gòu)建與轉(zhuǎn)染效率分析本實驗通過麻省理工學院的CRISPR Design (http://crispr.Mit.edu)軟件設(shè)計4對長20nt的gRNA,以gRNA-T2為模板進行PCR,擴增得到完整的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載體pCMV-DsRed導入絨山羊胎兒成纖維細胞中,通過流式細胞儀進行分析的結(jié)果表明,最優(yōu)轉(zhuǎn)染條件為：每1×106個絨山羊胎兒成纖維細胞加入pCMV-DsRed質(zhì)粒10μg(1μg/μL), Opti-MEM 90μL,電壓225V,脈沖時間2.5ms。利用以上最佳轉(zhuǎn)染條件將hCas9載體和gRNA共同導入阿爾巴斯絨山羊胎兒成纖維細胞中,培養(yǎng)48h后提取基因組,設(shè)計跨打靶位點的PCR引物進行PCR擴增,使用Surveyor突變檢測試劑盒進行檢測,確定有3個gRNA敲除效率較高,可以進行下一步實驗。2、利用CRISPR-Cas9技術(shù)制備MSTN基因敲除絨山羊胎兒成纖維細胞和肌肉衛(wèi)星細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 導入阿爾巴斯絨山羊胎兒成纖維細胞中和肌肉衛(wèi)星細胞中,隨機挑取單個細胞培養(yǎng),建立單克隆細胞系。對每個單克隆細胞系的基因組進行PCR擴增并進行測序,篩選出靶位點發(fā)生突變的單克隆細胞系。本研究共獲得62個胎兒成纖維單克隆細胞系,其中有10個單等位基因敲除的單克隆細胞系和10個雙等位基因敲除的單克隆細胞系,總敲除效率為32.25%。還獲得了6個肌肉衛(wèi)星單克隆細胞系,其中有5個雙等位基因敲除的陽性單克隆細胞系,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胞細胞系C205,通過qPCR對MSTN基因敲除后的肌發(fā)育相關(guān)基因表達水平進行檢測,發(fā)現(xiàn)該細胞中MSTN基因mRNA表達量下降73.79%,Western blot的檢測結(jié)果也證實MSTN雙等位基因敲除細胞中MSTN蛋白的表達比野生型細胞MSTN蛋白的表達量減少。而隨著MSTN的表達的降低,MyoG、Myf5和Myf6等基因轉(zhuǎn)錄水平都有不同程度的上升,分別為對照細胞的1.53倍、5.68倍和3.683倍,說明MSTN對這三個基因起著負調(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.
【學位授予單位】：內(nèi)蒙古大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S827;Q78

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本文編號：1559801