CRISPR-Cas9介导FGF5敲除及Noggin过表达对毛发生长相关因子影响
发布时间：2018-09-18 13:45
【摘要】：基因编辑技术,兴起于上世纪80年代,开启了人1门对基因功能研究的热潮。CRISPR-Cas9作为近年来新兴的基因编辑技术,具有载体构建简单、基因敲除率高等优势。绒山羊作为我国独特的生物资源,具有产绒量高,绒质好等诸多优点,其羊绒更具有“生物黄金”的美誉。因此进行高产绒量的品种选育具有极高的意义和巨大的市场潜力。而毛发生长涉及到一个及其复杂的基因调控机制。以往研究表明,Noggin基因的过表达和FGF5基因的敲除都会促进毛发生长。本实验主要针对毛发生长正调控基因Noggin和负调控基因FGF5,利用CRISPR-Cas9技术和基因随机整合技术,研究CRISPR-Cas9的敲除效率及FGF5敲除Noggin基因过表达同时作用后,在转录水平上检测其对毛发生长相关基因的mRNA表达量,从而预测FGF5、Noggin双基因编辑对毛发生长的影响。1、小鼠胎儿成纤维细胞转染条件优化本实验利用电转染法,以pCMV-DsRed质粒质量梯度和电转染电压和脉冲时间组合参数梯度,研究了转染小鼠胎儿成纤维细胞的最佳转染条件,结果显示电转染质粒总质量、电压、脉冲时间的最佳组合为10μg/160V/5mM。2、gRNA-FGF5、Noggin随机整合载体pK14-Noggin和Noggin定点整合载体pKI-Noggin的构建本研究成功的构建了针对小鼠FGF5基因的向导RNA(gRNA-FGF5),结果显示其可成功引导Cas9蛋白到目的靶位点进行双链DNA切割。本实验同时成功构建了两个Noggin基因皮肤特异性表达载体。一个为Noggin随机整合载体pK14-Noggin,用于Noggin过表达研究。另一个为Noggin定点整合载体pKI-Noggin,用于今后CRISPR/cas9,gRNA-FGF5介导Noggin定点整合模式动物构建。3、FGF5基因敲除效率和Noggin基因随机整合检测我们对以上实验构建的gRNA-FGF5和Noggin基因皮肤特异性随机整合载体pK14-Noggin进行效率检测。首先,我们通过电转染的方法将Cas9质粒与gRNA-FGF5共同转染小鼠胎儿成纤维细胞,提取细胞总基因组作为模板,用FGF5靶位点检测引物进行PCR反应,并利用Surveyor突变检测试剂盒检测,结果证明实验设计的gRNA-FGF5可以高效的引导Cas9蛋白对目标靶点的切割。本研究还运用T-A克隆的方法将PCR产物连入pMD19-T载体,随机挑取20个阳性重组子委托北京华大基因公司进行测序。结果显示：随机挑取的20个重组子中有11个在靶位点具有不同程度的碱基缺失与插入,由此结论获得突变率约为55%。其次,我们将pK14-Noggin质粒电转染小鼠胎儿成纤维细胞,48h后,提取细胞总蛋白,利用western blot检测,结果显示Noggin蛋白相对表达量是相同来源及代次野生型对照细胞的1.41倍。4、FGF5敲除Noggin过表达对毛发生长相关因子的影响本实验将Cas9、gRNA-FGF5转染MEF细胞得到EG1; pK14-Noggin转染小鼠MEF细胞得到EG2; Cas9、gRNA-FGF5、pK14-Noggin共转染小鼠MEF细胞得到EG3,并以未转基因的小鼠MEF细胞CG作为对照。转染48h后,以4种细胞的总cDNA作为模板,利用qPCR的方法,在mRNA水平上检测Tβ4、β-catenin、 vegf三个基因的mRNA相对表达量,结果显示：FGF5敲除的细胞β-catenin、 Vegf、Tβ4mRNA相对表达量分别是空白对照的2.82/1.37/1.47倍。noggin随机整合的细胞β-catenin、Vegf、Tβ4 mRNA相对表达量分别是空白对照的2.44/1.32/1.37倍 。FGF5敲除、noggin随机整合的细胞β-catenin、 Vegf、Tβ4 mRNA相对表达量分别是空白对照的3.22/1.69/1.71倍。
[Abstract]:Gene editing technology, rising in the 1980s, has opened a new upsurge in gene function research. As a new gene editing technology in recent years, CRISPR-Cas9 has the advantages of simple vector construction and high gene knockout rate. As a unique biological resource in China, cashmere goats have many advantages, such as high cashmere yield, good cashmere quality and so on. Therefore, it is of great significance and great market potential to breed varieties with high cashmere yield. Hair growth involves a complicated gene regulation mechanism. Previous studies have shown that overexpression of Noggin gene and knockout of FGF5 gene can promote hair growth. The knockout efficiency of CRISPR-Cas9 and the over-expression of Noggin gene knocked out by FGF5 were studied by using CRISPR-Cas9 technique and random integration technique. The mRNA expression of genes related to hair growth was detected at transcriptional level to predict the editorial pairs of FGF5 and Noggin genes. Effects of Hair Growth on the Transfection Conditions of Mouse Fetal Fibroblasts The optimal combination was 10 ug/160V/5mM.2, gRNA-FGF5, Noggin random integration vector pK14-Noggin and Noggin site-directed integration vector pKI-Noggin. In this study, we successfully constructed a guided RNA (gRNA-FGF5) targeting mouse fibroblast growth factor 5 gene. The results showed that it could successfully guide Cas9 protein to target sites for double stranded DNA cleavage. Two Noggin gene skin-specific expression vectors were successfully constructed. One was a Noggin random integration vector pK14-Noggin for Noggin overexpression study. The other was a Noggin site-specific integration vector pKI-Noggin for future CRISPR/cas9. GRNA-FGF5 mediated Noggin site-specific integration model animal construction. The efficiency of gRNA-FGF5 and Noggin gene skin-specific random integrative vector pK14-Noggin constructed in the above experiments was tested by machine integration test. First, we transfected the cas9 plasmid and gRNA-FGF5 into mouse fetal fibroblasts by electroporation, extracted the total genome of the cells as a template, and detected the primers with the target site of FGF5. The results of PCR reaction and Surveyor mutation detection kit showed that the designed gRNA-FGF5 could efficiently guide the cleavage of Cas9 protein to target sites. In this study, the PCR products were linked to pMD19-T vector by T-A cloning, and 20 positive recombinants were randomly selected for sequencing by Beijing Huada Gene Company. The results showed that 11 of the 20 recombinants were deleted and inserted at different levels at the target site, and the mutation rate was about 55%. Secondly, we transfected pK14-Noggin plasmid into mouse fetal fibroblasts. After 48 hours, the total cell protein was extracted and detected by Western blot. The results showed that Noggin protein was relative. In this study, EG1 was obtained by transfecting Cas9, gRNA-FGF5 into MEF cells, EG2 was obtained by transfecting pK14-Noggin into MEF cells, EG3 was obtained by co-transfecting mouse MEF cells with Cas9, gRNA-FGF5 and pK14-Noggin, and EG3 was obtained by co-transfecting mouse MEF cells with pK14-Noggin. After 48 hours of transfection, the relative mRNA expression levels of T-beta-4, beta-catenin and VEGF genes were detected by q-PCR. The results showed that the relative mRNA expression levels of beta-catenin, Vegf and T-beta-4 in the knockout cells were 2.82/1.37/1.47 times higher than those in the blank control. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 2.44/1.32/1.37 times higher than that in blank control cells, respectively. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 3.22/1.69/1.71 times higher than that in blank control cells.
【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】：S852.2
本文编号：2248105
[Abstract]:Gene editing technology, rising in the 1980s, has opened a new upsurge in gene function research. As a new gene editing technology in recent years, CRISPR-Cas9 has the advantages of simple vector construction and high gene knockout rate. As a unique biological resource in China, cashmere goats have many advantages, such as high cashmere yield, good cashmere quality and so on. Therefore, it is of great significance and great market potential to breed varieties with high cashmere yield. Hair growth involves a complicated gene regulation mechanism. Previous studies have shown that overexpression of Noggin gene and knockout of FGF5 gene can promote hair growth. The knockout efficiency of CRISPR-Cas9 and the over-expression of Noggin gene knocked out by FGF5 were studied by using CRISPR-Cas9 technique and random integration technique. The mRNA expression of genes related to hair growth was detected at transcriptional level to predict the editorial pairs of FGF5 and Noggin genes. Effects of Hair Growth on the Transfection Conditions of Mouse Fetal Fibroblasts The optimal combination was 10 ug/160V/5mM.2, gRNA-FGF5, Noggin random integration vector pK14-Noggin and Noggin site-directed integration vector pKI-Noggin. In this study, we successfully constructed a guided RNA (gRNA-FGF5) targeting mouse fibroblast growth factor 5 gene. The results showed that it could successfully guide Cas9 protein to target sites for double stranded DNA cleavage. Two Noggin gene skin-specific expression vectors were successfully constructed. One was a Noggin random integration vector pK14-Noggin for Noggin overexpression study. The other was a Noggin site-specific integration vector pKI-Noggin for future CRISPR/cas9. GRNA-FGF5 mediated Noggin site-specific integration model animal construction. The efficiency of gRNA-FGF5 and Noggin gene skin-specific random integrative vector pK14-Noggin constructed in the above experiments was tested by machine integration test. First, we transfected the cas9 plasmid and gRNA-FGF5 into mouse fetal fibroblasts by electroporation, extracted the total genome of the cells as a template, and detected the primers with the target site of FGF5. The results of PCR reaction and Surveyor mutation detection kit showed that the designed gRNA-FGF5 could efficiently guide the cleavage of Cas9 protein to target sites. In this study, the PCR products were linked to pMD19-T vector by T-A cloning, and 20 positive recombinants were randomly selected for sequencing by Beijing Huada Gene Company. The results showed that 11 of the 20 recombinants were deleted and inserted at different levels at the target site, and the mutation rate was about 55%. Secondly, we transfected pK14-Noggin plasmid into mouse fetal fibroblasts. After 48 hours, the total cell protein was extracted and detected by Western blot. The results showed that Noggin protein was relative. In this study, EG1 was obtained by transfecting Cas9, gRNA-FGF5 into MEF cells, EG2 was obtained by transfecting pK14-Noggin into MEF cells, EG3 was obtained by co-transfecting mouse MEF cells with Cas9, gRNA-FGF5 and pK14-Noggin, and EG3 was obtained by co-transfecting mouse MEF cells with pK14-Noggin. After 48 hours of transfection, the relative mRNA expression levels of T-beta-4, beta-catenin and VEGF genes were detected by q-PCR. The results showed that the relative mRNA expression levels of beta-catenin, Vegf and T-beta-4 in the knockout cells were 2.82/1.37/1.47 times higher than those in the blank control. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 2.44/1.32/1.37 times higher than that in blank control cells, respectively. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 3.22/1.69/1.71 times higher than that in blank control cells.
【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】：S852.2
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