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鱼类电脉冲基因转移方法的建立
谢岳峰
Subtype硕士
Thesis Advisor朱作言
1990
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Abstract以电脉冲方法成功地将外源基因导入泥鳅和鲫鱼的受精卵,并获得了整合外源基因的转基因鱼。在最适宜条件下,该方法的基因转移率达62.5%,受体鱼每细胞携带的外源基因可超过100拷贝。用自制的电脉冲系统,研究了鱼类受精卵对电脉冲的耐受能力,对于幅度为250伏特的电脉冲,泥鳅受精卵在放电时间小于6.6毫秒的范围内可以正常发育,鲫鱼受精卵在放电时间小于2.4毫秒时胚胎发育不受影响。泥鳅受精卵转移pMhGH重组DNA后在不同发育时期制备胚胎DNA,进行斑点杂交,发现外源基因异入受精卵后在原肠胚前进行扩增,随后开始缓慢降解。用不同脉冲条件对泥鳅和鲫鱼受精卵进行基因转移,检测1月龄的泥鳅和3月龄的鲫鱼,结果表明外源基因转移率及其拷贝数与电脉冲宽度和幅度,电荷量等均呈正相关关系。用环状或线性的外源基因对鲫鱼受精卵进行转移,二者的转移率没有显著差异,随着接受处理的受精卵受精后时间的延后,外源基因导入率呈下降趋势。对转基因鲫鱼的鳍条组织DNA进行限制性酶切消化及Southern吸印转移杂交,证明导入的线型基因已整合到受体鱼的染色体组中,其中部分外源基因受到修饰,导入的不状基因可能整合于染色体组中,也可能以游离的大分子多聚体形式存在。
Other AbstractForeign genes were successfully introduced into the fertilized eggs of fish via electroporation, and transgenic fishes integrated with foreign gene were obtained. The transfer efficience (TE) was up to 62.5% in the optimum condition, well compares with the results of microinjection method of gene transfer. The toleration of the fish fertilized eggs against electroporation was tested using a capacitor-based electroporator. The embryogenesis of loach and gold fish were not hindered by electroporation when it's voltage was set at 250V and it's width (duration) were below 6.6ms and 2.4ms respectively. Dot blot analysis of embryonic DNA showed that the replication of the foreign gene (pMhGH) in host loach eggs was accelerated from blastula stage to gastrula stage, then decelerated. Both total DNA of 1-month-old loach and 3-month-old gold fish were dot blotted to detect the persistence of foreign gene. The TE and the persisted copy number (PCN) were positively correlated with voltage, width and electric quantity of pulse, and negatively with the lag between fertilization and electroporation. There were not significant difference between the TE and PCN of circular pMhGH and linear pMhGH/H when transferred into eggs of gold fish. The integration of the introduced linear pMhGH/H not circular pMhGH, was surely demonstrated by the restriction enzyme digestion followed by Southern blot of total DNA extracted from transgenic gold fish.
Pages38
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12816
Collection学位论文
Recommended Citation
GB/T 7714
谢岳峰. 鱼类电脉冲基因转移方法的建立[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1990.
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