本文選題：轉(zhuǎn)基因小鼠 + SOD2��； 參考：《大理大學(xué)》2017年碩士論文

【摘要】：目的:熟悉基因組編輯核酸酶三大類之一的Talens,成功利用Talens系統(tǒng)構(gòu)建SOD2轉(zhuǎn)基因小鼠模型,并通過繁殖建系以及后期的鑒定獲得SOD2換基因小鼠的純合子模型。方法:(1)用野生型C57小黑鼠作為實(shí)驗(yàn)對(duì)象,在Talens系統(tǒng)的作用下構(gòu)建SOD2轉(zhuǎn)基因小鼠模型,并利用常規(guī)PCR和1%瓊脂糖凝膠電泳等系統(tǒng)進(jìn)行驗(yàn)證。(2)將構(gòu)建的雜合子SOD2轉(zhuǎn)基因小鼠模型與野生型C57小鼠進(jìn)行繁殖,通過常規(guī)PCR和1%瓊脂糖凝膠電泳等系統(tǒng)篩選出表達(dá)陽性的小鼠,并使用陽性小鼠繼續(xù)與野生型C57小鼠進(jìn)行繁殖建系。(3)待各品系達(dá)到一定規(guī)模后,每組選兩只陽性結(jié)果小鼠并用野生型C57小黑鼠做對(duì)照,利用Wb和Q-PCR篩選出兩個(gè)更適合進(jìn)行回交的品系,然后進(jìn)行同胞回交直到獲得純合子的SOD2轉(zhuǎn)基因小鼠。結(jié)果:(1)通過常規(guī)PCR和1%瓊脂糖凝膠電泳等系統(tǒng),成功獲得雜合子的SOD2轉(zhuǎn)基因小黑鼠10只。(2)通過繁殖最終成功獲得符合回交要求的1號(hào)、2號(hào)、3號(hào)、4號(hào)、7號(hào)、9號(hào)6個(gè)品系,各品系分別有8只、10只、9只、10只、8只、9只陽性結(jié)果小鼠。(3)6個(gè)品系的陽性小鼠數(shù)量都在10只左右,6個(gè)品系全部符合小鼠回交要求,通過做SOD2 Q-PCR獲得結(jié)果,6個(gè)品系中7號(hào)品系最適合同胞回交,同胞回交最終7號(hào)品系都獲得了純合子的SOD2轉(zhuǎn)基因小鼠。結(jié)論:(1)通過Talens系統(tǒng)能夠完成SOD2轉(zhuǎn)基因小鼠模型的構(gòu)建。(2)通過正常的繁殖建系,再配合上常規(guī)PCR。1%瓊脂糖凝膠電泳、免疫印跡、Q-PCR等技術(shù)可以完成對(duì)SOD2轉(zhuǎn)基因小鼠基因型的鑒定,從而給SOD2轉(zhuǎn)基因小鼠的回交獲得純合子打下了技術(shù)層面的基礎(chǔ),最終獲得了穩(wěn)定遺傳的SOD2轉(zhuǎn)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.
【學(xué)位授予單位】：大理大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R-332

【參考文獻(xiàn)】

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1 ;Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro[J];World Journal of Gastroenterology;2008年15期

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本文編號(hào)：1805140