利用Red重组系统快速构建G蛋白偶联受体基因打靶载体

发布时间：2018-05-11 17:29

本文选题：基因敲除 + 打靶载体构建　；参考：《华东师范大学》2011年硕士论文

【摘要】：G蛋白偶联受体(GPCR)是一类具有七次跨膜结构域的受体超家族,占人类基因组的3%-4%,具有广泛的生理功能,并且是最重要的药物作用靶点。粘附亚家族成员绝大多数为功能不明、配体未知的孤儿受体。阐明该家族成员的生理功能对于基础理论研究和潜在药物靶点的发现具有十分重大的意义。 GPCR粘附家族是一类非常独特的GPCRs亚家族,它是GPCRs家族中唯一具有与其他膜蛋白家族(例如酪氨酸激酶受体钙粘素受体等)相类似长的N末端结构的成员,很明显对于G蛋白粘附家族的研究还需要很多工作要完成。所以我们设计了GPCR粘附家族成员中Gpr56, Bail, Gpr111的完全基因敲除打靶载体以及Gpr126的条件性基因敲除载体,用来研究上述基因的功能。基因敲除小鼠模型是在动物体内研究基因功能的最重要和可靠的手段之一。由于PCR、酶切和连接长片段目的序列的难度,利用常规的分子克隆的方法构建基因敲除打靶载体一般耗时较长,甚至无法完成特定基因打靶载体的构建。基因敲除技术包括完全敲除基因打靶和条件性敲除基因打靶技术。本研究合理应用Red重组系统,通过两次重组,实现完全基因敲除打靶载体的快速构建。根据多次反复实验,改进了利用Red重组系统构建条件性基因敲除打靶载体构建的方法,能够将两个LoxP位点更加准确的插入到目标位置。
[Abstract]:G protein-coupled receptor (GPCR) is a kind of receptor superfamily with seven transmembrane domains, which accounts for 3- 4 of the human genome. It has a wide range of physiological functions and is the most important target of drug action. Most of the adherent subfamily members are orphan receptors with unknown function and unknown ligand. It is of great significance to clarify the physiological function of the family members for basic theoretical research and discovery of potential drug targets. The GPCR adhesion family is a very unique subfamily of GPCRs. It is the only member of the GPCRs family that has a long N-terminal structure similar to that of other membrane proteins (such as tyrosine kinase receptor cadherin receptor, etc.). It is clear that there is still much work to be done on the G-protein adhesion family. So we designed the complete gene knockout vector of Gpr56, Bailand Gpr111 and the conditional gene knockout vector of Gpr126 to study the function of these genes. Knockout mouse model is one of the most important and reliable methods to study gene function in animals. Because of the difficulty of PCR, enzyme digestion and ligation of long fragment target sequence, the construction of gene knockout targeting vector by conventional molecular cloning method is usually time-consuming, and even the construction of specific gene targeting vector can not be completed. Gene knockout techniques include complete knockout and conditional knockout. In this study, the Red recombination system was used to construct the complete gene knockout vector by twice recombination. According to repeated experiments, the method of constructing conditional gene knockout vector using Red recombination system is improved, and two LoxP sites can be inserted more accurately into the target position.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R3416

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