FGF5基因敲除辽宁绒山羊胎儿成纤维细胞系的构建

发布时间：2018-03-12 08:35

本文选题：FGF5　切入点：辽宁绒山羊　出处：《吉林农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：成纤维细胞生长因子5(Fibroblast growth factors 5,FGF5)是影响毛囊周期性生长的重要生长因子。本研究利用CRISPR/Cas9系统对辽宁绒山羊FGF5基因进行敲除,为深入研究FGF5的功能奠定基础。根据Genebank中已发表山羊FGF5(KC236981.1)基因编码区的序列信息设计引物,扩增出辽宁绒山羊FGF5基因的完整CDs区(Coding sequence)813bp,测序结果并未发现其剪接体FGF5s。FGF5基因中CDs区共编码270个氨基酸,理论分子质量为29.55kDa,理论等电位点为10.59,为亲水性蛋白,有信号肽,属于分泌蛋白,经序列对比分析其与牛的同源性为97.2%。采用组织块贴壁法分离辽宁绒山羊胎儿成纤维细胞,对其进行性别鉴定、生长曲线与核型分析,选出生长旺盛和核型正常的胎儿成纤维细胞用于后续研究。结果显示,成功建立了辽宁绒山羊胎儿成纤维细胞的体外培养体系,细胞系S1为雌性,S2为雄性,其生长旺盛,状态良好,核型正常,有30对染色体。利用生物学软件,针对绒山羊FGF5基因第一外显子,设计5个预测的sgRNA靶位点。构建CRISPR/Cas9重组载体,分别为PX459-FGF5-l、PX459-FGF5-2、PX459-FGF5-3、PX459-FGF5-4和PX459-FGF5-5,经测序表明载体连接成功。采用脂质体3000介导5个PX459-FGF5载体分别转染辽宁绒山羊胎儿成纤维细胞,经过T7EI酶酶切检测靶序列的有效性,选出敲除效率好载体转染的细胞,单克隆细胞扩繁培养,再提取基因组,脱靶检测,PCR及克隆测序验证。结果表明,有2个PX459-FGF5载体成功地进行了特异性切割和突变。随机选取60个单克隆细胞,测序获得23株辽宁绒山羊成纤维转基因细胞系(包括缺失插入和替换),均在靶位点发生FGF5突变,总突变率为38.3%。其中有9株发生插入缺失的单克隆细胞系适于作为体细胞核移植的供体细胞,为高产绒量辽宁绒山羊新品种的培育奠定了基础。
[Abstract]:Fibroblast growth factors 5 (FGF5) is an important growth factor affecting the periodic growth of hair follicles. In this study, Liaoning Cashmere Goat FGF5 gene was knockout by CRISPR/Cas9 system. In order to further study the function of FGF5, primers were designed according to the sequence information of goat FGF5 (KC236981.1) gene coding region published in Genebank. The complete CDs sequence sequence of Liaoning Cashmere goat FGF5 gene was amplified. The results of sequencing did not show that the CDs region of the splice FGF5s.FGF5 gene encodes 270 amino acids, the theoretical molecular weight is 29.55 kDa, and the theoretical equipotential point is 10.59, which is a hydrophilic protein with a signal peptide. It belongs to secretory protein, and its homology with cattle is 97.2.The fetal fibroblasts of Liaoning cashmere goat were separated by tissue block adhesion method, and their sex identification, growth curve and karyotype analysis were carried out. Fetal fibroblasts with strong growth and normal karyotype were selected for further study. The results showed that the in vitro culture system of Liaoning cashmere goat fetal fibroblasts was successfully established. The cell line S1 was female and S 2 was male and its growth was vigorous. In good condition, normal karyotype and 30 pairs of chromosomes, five predicted sgRNA target sites were designed for the first exon of FGF5 gene in cashmere goats by using biological software. CRISPR/Cas9 recombinant vector was constructed. They were PX459-FGF5-2, PX459-FGF5-3, PX459-FGF5-4 and PX459-FGF5-5, respectively. The ligation of PX459-FGF5-4 and PX459-FGF5-5 showed that the five PX459-FGF5 vectors were transfected into Liaoning cashmere goat fetal fibroblasts by liposome 3000, and the target sequences were detected by T7EI enzyme digestion. The transfected cells with good knockout efficiency vector were selected, and the monoclonal cells were propagated and cultured. Then the genome was extracted, and the PCR and clone sequencing were carried out. The results showed that, Two PX459-FGF5 vectors were successfully dissected and mutated. Twenty three Liaoning Cashmere Goat fibroblast transgenic cell lines (including deletion insertion and substitution) were sequenced from 60 monoclonal cells. All of them had FGF5 mutations at the target sites. The total mutation rate was 38.3. Among them, 9 clones with insertion deletion were suitable for donor cells of somatic nuclear transfer, which laid a foundation for the breeding of new varieties of Liaoning cashmere goat with high yield.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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