CRISRCas9介导DGAT1基因在小鼠MSTN位点定点整合研究.docx

优秀毕业论文 精品参考文献资料 内蒙古大学硕士学位论文本实验采取了电转染和脂质体转染两种方式对两种不同的细胞进行转染比 内蒙古大学硕士学位论文 本实验采取了电转染和脂质体转染两种方式对两种不同的细胞进行转染比 较，实验结果发现，对于小鼠成纤维细胞电转染的效率明显高于脂质体转染。而 小鼠胚胎干细胞用脂质体转染的效果明显好于电转。 3对转DGATl基因细胞的检测 通过跨同源臂检测的方法证明DGATl基因己定点整合到细胞的MSTN位点。 为了验证基因插入后的效果，通过实时定量PCR分别检测了目的基因及脂肪代谢 相关基因和与肌肉发育相关基因的表达情况。结果表明，转阳性细胞中DGATl 的表达量是对照组的32．76倍。随着DGATl含量的提高，脂肪代谢有关基因的表 达有不同程度的上调，乙酰辅酶A羧化酶上调1．58倍，脂肪酸结合蛋白4上调7．1 1 倍，DGAT2上调2．93倍。说明DGATl的上调对促进了脂肪的合成。解偶联蛋白l 表达量也提高了2．17倍。因为在小鼠MSTN基因上定点插入DGATl，所以本实验 还检测了肌肉相关基因的表达情况，结果显示Foxol相比对照上调23．53倍。Mef2c 是对照的O．66倍，表达量下调。 关键词：CRISPR／Cas9；MSTN；DGATl；基因打靶 万方数据 CRISPR／ CRISPR／Cas9介导DGATl基因在小鼠MSTN位点定点整合研究 CRISPR／CAS9 MEDIATED DGAT 1 GENE KNoCK．IN AT MSTN LoCUS IN MoUSE AB STRACT MSTN(Myostatin)is the negative regulator in skeletal muscle growth，the MSTN gene knock—out could enhance muscle mass in animal models．DGAT 1 is a glycerol acyltransferase，the enzyme is closely related to fat metabolism and lipid deposition in tissues．CRISPR／Cas9 recognise the DNA specific sites by RNA，the core RNA-guided Cas9 endonuclease in the DNA has been hamessed to realize gene mutation，DNA deletion and insertion．The objective of this study is to get MSTN knock··out and DGAT 1 gene knock·-in in mouse embryonic fibroblast cells by CRISPR／Cas9 technology．The results will provide experimental bases for the production of animals． The results are as follow： l The gRNA in CRISPR／Cas9 system and knock—in vector CAG．DGAT 1 were successfully constructed gRNA vector was successfully constructed for MSTN in mouse，The results of surveyor mutation detection proved that Cas9 call efficiently cut DNA at specific sites under the guidance of the gRNA．DGAT l gene knock—in vector 3 万方数据 内蒙古大学硕士学位论文was 内蒙古大学硕士学位论文 was proved correct by PCR，enzyme digestion and sequencing． 1．The improvement of mouse fibroblast cells and mouse embryonic stem cells transfected selection Electroportion and lipofectin transfection were compared in two different cells．The results showed that electroportion was significantly higher than lipo