使用TALEN技术构建的GPR敲出小鼠的筛选鉴定.docVIP

PAGE 湖南大学毕业论文（设计） 第PAGE I页 HUNAN UNIVERSITY 毕业设计(论文) 设计论文题目： 使用TALEN技术构建的GPR3 敲除小鼠的筛选鉴定 学生姓名： 唐开翼 学生学号： 20090940219 专业班级： 生物技术2009级2班 学院名称： 生物学院 指导老师： 廖红东 张儒 学院院长： 谭蔚泓 2013年 6月 日 使用TALEN技术构建的GPR3敲除小鼠的筛选鉴定 摘 要 TALEN(transcription activator-like effector nuclease)技术是一种源于植物致病菌Xanthomonas的崭新的靶向基因操作技术。与传统方法相比，TALEN具有很高的打靶效率，一般在60%左右，甚至更高，大大减少了构建基因敲除动物模型的工作量。GPR3编码产物是七次跨膜G蛋白偶联受体（G-protein coupled receptor，GPCR），前期研究证实，GPR3受体与神经退行性疾病和减数分裂阻遏等密切相关，然而其结构与功能的研究尚处于起步阶段，且作用于它的内源性激动剂和拮抗剂至今仍不清楚。本实验设计旨在综合运用鼠尾基因组DNA提取、PCR、酶切、DNA电泳和TA克隆测序等一整套现代分子生物学技术，筛选出用TALEN技术构建的首代GPR3敲除阳性小鼠，为研究GPR3受体的在体生理功能及其调控机制提供可靠的动物模型，同时也为以GPR3受体为靶点的新药设计提供正确的药物筛选模型。 关键词：基因敲除， TALEN，受体， GPR3，小鼠 Screening and identification of the first generation of GPR3 knockout mouse generated by TALEN Abstract TALE nuclease (TALEN) is a new technology to specifically and effectively modify genome. Transcription activator-like effectors (tales) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas. Compared with traditional gene knockout methods, TALEN has a higher targeting efficiency, generally around 60%. It greatly reduces the workload of establishing a gene-knockout animal model. The gene GPR3 encodes a G protein-coupled receptor 3 (GPR3) which is a novel seven ?-helix trans-membrane receptors and an orphan receptor. Previous researches have identified?that GPR3 plays an important pole in promoting neurite outgrowth，maintaining meiotic arrest and aggravating Alzheimer’s disease. However, the signal transduction and its functional regulation mechanisms of GPR3 is still unclear. By using a series of molecular biology technology such as genomic DNA isolation from mouse tails, enzymatic restriction digestion, PCR, DNA agrose-gel electrophoresis, TA cloning and DNA sequencing, we?screened the first generation of GPR3 knockout mouse generated by embryo microinjection of TALEN plasmids targeting GPR3. The correct gene-knockout