CRISPR-Cas9技术介导阿尔巴斯白绒山羊MSTN基因敲除的研究

发布时间：2018-03-03 06:00

本文选题：肌肉生长抑制素　切入点：CRISPR-Cas9　出处：《内蒙古大学》2015年硕士论文　论文类型：学位论文

【摘要】：肌肉生长抑制素(Myostatin, MSTN)又称生长分化因子-8 (growth differentiation factor 8, GDF-8),是一类重要的骨骼肌细胞生长发育的负调控因子,研究表明,该基因的缺失、突变能加速肌肉生长和改变红白肌的组成比例并增加肌肉量。CRISPR-Cas系统是一种来源于细菌获得性免疫的由RNA指导Cas蛋白对靶向基因进行修饰的技术。本研究利用CRISPR-Cas9系统对阿尔巴斯白绒山羊胎儿成纤维细胞和肌肉卫星细胞中的MSTN基因进行了敲除,并通过实时定量PCR与Western blot检测了MSTN基因敲除后成纤维细胞中与之相关基因的表达情况,为通过CRISPR-Cas9系统生产基因编辑动物奠定基础。1、gRNA表达载体的设计、构建与转染效率分析本实验通过麻省理工学院的CRISPR Design (http://crispr.Mit.edu)软件设计4对长20nt的gRNA,以gRNA-T2为模板进行PCR,扩增得到完整的gRNA(最终PCR产物长度为455bp)。为了筛选适应于山羊细胞的最优转染条件,本研究利用电穿孔法将红色荧光蛋白表达载体pCMV-DsRed导入绒山羊胎儿成纤维细胞中,通过流式细胞仪进行分析的结果表明,最优转染条件为：每1×106个绒山羊胎儿成纤维细胞加入pCMV-DsRed质粒10μg(1μg/μL), Opti-MEM 90μL,电压225V,脉冲时间2.5ms。利用以上最佳转染条件将hCas9载体和gRNA共同导入阿尔巴斯绒山羊胎儿成纤维细胞中,培养48h后提取基因组,设计跨打靶位点的PCR引物进行PCR扩增,使用Surveyor突变检测试剂盒进行检测,确定有3个gRNA敲除效率较高,可以进行下一步实验。2、利用CRISPR-Cas9技术制备MSTN基因敲除绒山羊胎儿成纤维细胞和肌肉卫星细胞的的制备与分析采用最适合的转染条件,将hCas9载体和gRNA 导入阿尔巴斯绒山羊胎儿成纤维细胞中和肌肉卫星细胞中,随机挑取单个细胞培养,建立单克隆细胞系。对每个单克隆细胞系的基因组进行PCR扩增并进行测序,筛选出靶位点发生突变的单克隆细胞系。本研究共获得62个胎儿成纤维单克隆细胞系,其中有10个单等位基因敲除的单克隆细胞系和10个双等位基因敲除的单克隆细胞系,总敲除效率为32.25%。还获得了6个肌肉卫星单克隆细胞系,其中有5个双等位基因敲除的阳性单克隆细胞系,总敲除效率为83.33%。本研究选取了双等位基因敲除的阳性成纤维细胞细胞系C205,通过qPCR对MSTN基因敲除后的肌发育相关基因表达水平进行检测,发现该细胞中MSTN基因mRNA表达量下降73.79%,Western blot的检测结果也证实MSTN双等位基因敲除细胞中MSTN蛋白的表达比野生型细胞MSTN蛋白的表达量减少。而随着MSTN的表达的降低,MyoG、Myf5和Myf6等基因转录水平都有不同程度的上升,分别为对照细胞的1.53倍、5.68倍和3.683倍,说明MSTN对这三个基因起着负调控作用。
[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.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827;Q78

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