SOD2小鼠模型的构建及鉴定

发布时间：2018-04-26 07:56

本文选题：转基因小鼠 + SOD2　；参考：《大理大学》2017年硕士论文

【摘要】：目的:熟悉基因组编辑核酸酶三大类之一的Talens,成功利用Talens系统构建SOD2转基因小鼠模型,并通过繁殖建系以及后期的鉴定获得SOD2换基因小鼠的纯合子模型。方法:(1)用野生型C57小黑鼠作为实验对象,在Talens系统的作用下构建SOD2转基因小鼠模型,并利用常规PCR和1%琼脂糖凝胶电泳等系统进行验证。(2)将构建的杂合子SOD2转基因小鼠模型与野生型C57小鼠进行繁殖,通过常规PCR和1%琼脂糖凝胶电泳等系统筛选出表达阳性的小鼠,并使用阳性小鼠继续与野生型C57小鼠进行繁殖建系。(3)待各品系达到一定规模后,每组选两只阳性结果小鼠并用野生型C57小黑鼠做对照,利用Wb和Q-PCR筛选出两个更适合进行回交的品系,然后进行同胞回交直到获得纯合子的SOD2转基因小鼠。结果:(1)通过常规PCR和1%琼脂糖凝胶电泳等系统,成功获得杂合子的SOD2转基因小黑鼠10只。(2)通过繁殖最终成功获得符合回交要求的1号、2号、3号、4号、7号、9号6个品系,各品系分别有8只、10只、9只、10只、8只、9只阳性结果小鼠。(3)6个品系的阳性小鼠数量都在10只左右,6个品系全部符合小鼠回交要求,通过做SOD2 Q-PCR获得结果,6个品系中7号品系最适合同胞回交,同胞回交最终7号品系都获得了纯合子的SOD2转基因小鼠。结论:(1)通过Talens系统能够完成SOD2转基因小鼠模型的构建。(2)通过正常的繁殖建系,再配合上常规PCR。1%琼脂糖凝胶电泳、免疫印迹、Q-PCR等技术可以完成对SOD2转基因小鼠基因型的鉴定,从而给SOD2转基因小鼠的回交获得纯合子打下了技术层面的基础,最终获得了稳定遗传的SOD2转基因小鼠。
[Abstract]:Objective: to familiarize Talens, one of the three genome-edited nucleases, to successfully construct the SOD2 transgenic mouse model by using Talens system, and to obtain the homozygote model of SOD2 transgenic mice by breeding and later identification. Methods using wild type C57 black mouse as experimental object, SOD2 transgenic mouse model was constructed with Talens system. The heterozygote SOD2 transgenic mice model was propagated with wild-type C57 mice by routine PCR and 1% agarose gel electrophoresis. The positive mice were screened by routine PCR and 1% agarose gel electrophoresis, and the positive mice were used to reproduce with wild type C57 mice. Two positive mice were selected from each group and the wild type C57 black mice were used as control. Two strains were screened by Wb and Q-PCR, and then sibling backcross was performed until the homozygous SOD2 transgenic mice were obtained. Results by routine PCR and 1% agarose gel electrophoresis, 10 SOD2 transgenic black mice with heterozygotes were successfully obtained by breeding, and 6 strains 1, 2, 3, 4, 7 and 9 were obtained by breeding. The number of positive mice of the 6 strains was about 10, and all the 6 strains met the requirement of backcross. The results obtained by SOD2 Q-PCR showed that line 7 was the most suitable for sibling backcrossing, and homozygous SOD2 transgenic mice were obtained in the final sibling backcross. Conclusion the construction of transgenic mouse model of SOD2 can be completed by Talens system. (2) normal breeding line, combined with conventional PCR.1% agarose gel electrophoresis and Western blotting Q-PCR, can be used to identify the genotypes of SOD2 transgenic mice. Thus, it laid a technical foundation for the backcross of SOD2 transgenic mice to obtain homozygote, and finally obtained stable inherited SOD2 transgenic mice.
【学位授予单位】：大理大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332

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