CRISPRCas9介导敲除内蒙古白绒羊胎儿成纤维细胞FGF5基因.docx

优秀毕业论文 精品参考文献资料 结果表明：通过生物信息学分析得到靶向于内蒙古白绒山羊基因组FGF5 结果表明：通过生物信息学分析得到靶向于内蒙古白绒山羊基因组FGF5 基因的79个潜在靶位点，从中选取了两个sgRNA序列，分别命名为FGF5．1 (AGAAGCGCCTCGCACCC A A A)和FGF5．2(CCCTGCCTCCTCCTCCTCCG)。 将两个sgRNA序列连接于pX330空载体，得到特异于白绒山羊基因组FGF5基 因的CRISPR／Cas9表达载体_px330—FGF5—1和pX330．FGF5-2。测序表明载 体构建正确。 利用pX330一FGF5．1和pX330一FGF5—2转染内蒙古白绒山羊胎儿成纤维细胞， Surveyor错配酶酶切和测序验证表明成功的在靶位点进行了特异性切割和突变。 利用流式细胞仪通过随机挑选得到24个单克隆细胞系，特异性扩增FGF5基因 片段，测序结果表明其中4个细胞系在靶位点发生FGF5基因突变。对这4个敲 除FGF5基因的阳性克隆细胞系进行潜在脱靶位点验证，均未发生基因突变。 综上所述，本文根据内蒙古白绒山羊FGF5基因的基因组序列设计特异性 sgRNA，构建CRISPR／Cas9表达载体，转染内蒙古白绒山羊胎儿成纤维细胞， 经过筛选与验证，得到4个定点敲除’FGF5基因的内蒙古白绒山羊胎儿成纤维细 胞的单克隆细胞系，为进一步利用克隆技术培育高产绒量内蒙古白绒山羊新品 系提供了条件。 关键词：成纤维细胞生长因子；CRISPR／Cas9技术；基因敲除；白绒山羊胎儿 成纤维细胞 万方数据 CRISPR／ CRISPR／Cas9 mediated FGF5 gene Knockout in Inner Mongolia Cashmere Goat Fetal Fibroblast ABSTRACT The Inner Mongolia Cashmere goat produces quaility cashmere，which e巧oys high reputation in the world．How to improve the cashmere production always been a important task of the cashmere goat breeding，molecular breeding provides a new method for breed improvement．The RNA mediated CRISPR／Cas9 system has been adapted as an effeicent genen-targeting technolgy,which include a non specific Cas9 nuclease and a set of programmable sequence specific guide RNA．The sequence specific RNA guide Cas9 to cleave DNA and generic double-strand breaks at target sites．Subsequent cellular DNA repair process leads to desired in sertions，deletions or substitutions at target sites． Fibroblast growth factor 5 is an important growth factor that affect the hair follicle cycle and hair growth and control the transformation of hair follicle from the growth stage to a resting phase．Knock-out of FGF5 gene promote the growth of cashrriere by deregulating the hair follicle growth cycle． In this study,we designed the specifics sglⅢA target to cashmere goat FGF5 gene and predicted the potential off-target sites of the sgmqA，then constructed CRISPR／Cas9 specific expression vectors target to cashmere goats FGF5 gene。The constructed