CRISPRCas9与TALENs介导奶山羊β酪蛋白位点基因打效率的比较研究.docx

优秀毕业论文 精品参考文献资料 囱苤直丕堂 囱苤直丕堂 塑主堂篁丝塞 共获得1 8株打靶细胞，pGlrkt定点整合的34株细胞系中获得11株打靶细胞，打靶效 率分别为26．47％和32．35％；在采用流式分选的方法得到23株TALENs介导的pFlrkt 定点整合细胞系中共获得5株打靶细胞，其打靶效率为21．74％；在铲取的细胞集落中 采用CRISPR／Cas9介导的pFlrkt载体定点整合的35株细胞系中获得26株打靶细胞， pGlrkt载体定点整合的27株细胞系中获得1 9株打靶细胞，其打靶效率分别为74．29％ 和70．37％；在采用流式分选的方法所得到的17株CRISPR／Cas9介导的pFlrkt定点整 合细胞系中共获得10株打靶细胞，其打靶效率为58．82％。实验证明，CRISPR／Cas9 介导的基因打靶效率要高于TALENs。 本实验最终获得59株办向如J『定点整合细胞系，这些细胞为今后的体细胞核移植、 胚胎移植提供了可选的供体细胞。获得这种敲除p_酪蛋白基因的奶山羊品种，能够提 高羊奶品质，降低羊奶加工难度。同时实验证明，在奶山羊上，CRISPR／Cas9介导的 基因打靶效率要高于TALENs。 关键词：CRISPR／Cas9；TALENs；p．酪蛋白(CSN2)；载体构建；基因打靶 万方数据 刘畅 刘畅 CRISPR／Cas9与TALENs介导羊奶山羊B一酪蛋白位点基因打靶效率的比较研究 TH’E STUDY oN EFFICIENCY OF CRISPR／Cas9 AND TALENs MEDlATED GENE T』蛾GETING AT DAIRY GoAT 13-CASE IN LOUC S Abstract CRISPR／Cas9 is a newly developed gene editing technology,which can introduce double strand break(DSB)by Cas9 that guided by sglⅢA to specific gene locus．Compared to ZFNs and TALENs，CRISPR／Cas9 not o由has a great advantage by avoiding the recognition DNA sequence by structural identification,but also more efficient and easier to operate and obtain homozygous mutants．It Can simultaneously introduce multiple mutations at different sites． In this study，a set of TALENs and a CRISPR／Cas9 plasmid were designed for editing the dairy goat fl-casein(CSN2)gene at exon2，which is the first coding exon． Two targeting donor plasmids aimed to the TALENs and CRISPR／Cas9 site，named pFlrkt and pGlrkt,weTe constructed on the basis of GFP—PGK-Neo‘plasmid．In the 1 024bp 5homologous arm was located at the upstream of CAGG promoter,and 1 028bp 3qaomologous a]nnl located at the downstream ofNeor gene．The exogenous gene Was hFat-1 and EGFP,respectively．nle structure ofthe recombinant plasmids Was verified by restriction fragment analysis and DNA sequencing． Next，to test the efficiency TALENs and CRISPR／Cas9 mediated gene knock-in， the EGFP and hfat一1 expression plasmids pFlrkt and pGlrkt were CO—transfected by electroporation to dairy goat fetal fibroblasts with TALENs and