CRISPRCas9系统介导羊MST基因敲除和定点整合fat1基因的研究.docx

优秀毕业论文 精品参考文献资料 CRISPR／ CRISPR／Cas9系统介导羊MSTN基因敲除和定点整合fat-I基因的研究 张驹 本实验使用电转法将线性化的fat一1载体、gRNA2和Cas9载体按比例转 入羊胎儿成纤维细胞中，通过流式细胞仪和口吸管2种方法挑取单克隆细胞。 共成功获得1 56株单克隆细胞，经过PCR和测序鉴定，其中在MSTN基因 位点定点整合fat．1基因的细胞系40株，外源基因整合效率为25．64％。 3、转基因绒山羊的制备 挑选l株阳性细胞通过体细胞核移植技术生产胚胎，共获得249枚胚胎， 选择其中的134枚克隆胚移植到56只受体羊中，成功获得1只转基因绒山羊， 其生长发育良好，经PCR初步鉴定在MSTN基因位点整合入fat．1外源基因。 关键词：CRISPR／Cas9；基因敲除；基因敲入；MSTN；fat一1 万方数据 内蒙古大学 内蒙古大学 硕士学位论文 GENERATION OF MSTN GENE KNOCK—OUT AND FAT一1 GENE KNOCK—IN GOAT VIA CRISPER／CAS9 AB STRACT The aims of genetic manipulation are to enhance agriculture by modifying domestic animal，to maximize yields and value．MSTN is known as a negative regulator of muscle development，mutations in the gene can lead to significantly increases in muscle development．Fat一1 gene encoding∞-3 PUFAs desaturase．can convea polyunsaturated fatty acids from∞一6 to∞一3．the increase of fat-1 gene is good for improving the content of∞-3 in animals．Our works is to construct gRNA expression vectors and new plasmid vectors for fat一1 gene targeting in MSTN gene locus，which enable knock—out MSTN gene and knock—in fat一1 gene at the same time．GEFs were transfected with those vectors，along with selecting the best CO—electroporation conditions by electroporation．Our method involved generating gene modified GEFs and using SCNT to generate transgenic goats which improve yield and quality of mutton． 1．Vector construction Two gRNA Vectors has been constructed successfully at MSTN of exon 1， the mutation effi ci ency of gRNA2 is higher than gRNA 1 through the survery 万方数据 cRJSP刚 cRJSP刚caS9系统介导羊MSTN基因敲除和定点整合fat一1基因的研究 张驹 enzyme mutation detection，SO gRNA2 is used as follow-up experiments； Sequencing confirmed targeting gene fragment was obtained and the fat一1 gene targeting vector in MSTN gene locus is constructed successfully． 2．Production of gene—modified monoclonal cells To screen the monoclonal cell lines by the methods of glass tube and flow cytometer,hCas9 vector，gRNA and donor plasmid were transfected into GFFs