睾丸内注射和体内电穿孔法建立转基因小鼠的实验研究

发布时间：2018-05-27 22:19

本文选题：辜丸内注射 + 体内电穿孔　；参考：《第一军医大学》2005年硕士论文

【摘要】：转基因动物在医学、生命科学、生物制药等领域有着广泛的研究和应用前景,因此科学家们一直在探索一种更简便易行、经济实用而又高效的转基因方法。近几年来,随着科学技术的发展,体内基因转移已成为用于基因治疗、生物学分析等领域非常受欢迎的一种技术,体内电穿孔技术就是其中之一。精原干细胞是精子的前体细胞,是雄性动物中唯一可以自我增殖、更新并向下一代传递遗传信息的细胞类型。对精原干细胞的操作和修饰在建立转基因动物模型、制备乳腺生物反应器等方面有重要意义。为了探讨将外源基因注射到在体睾丸生精小管内并进行体内电穿孔以建立转基因动物的可行性,我们首先设计了低压(30～110V/cm)和高压(500～900V/cm)两组不同的体内电穿孔参数对SPF级KM雄鼠睾丸进行在体电穿孔,以观察不同的电穿孔条件对SPF级KM雄鼠睾丸损伤程度和生殖能力的影响,从而筛选出合适的体内电穿孔参数。并在此基础上,我们对插入有增强型绿色荧光蛋白(EGFP)基因的真核表达质粒pCE-29DNA进行2种不同的处理:(1) pCE-29DNA+转染试剂(in vivo jetPI~(TM)+台盼蓝(TB);(2) pCE-29DNA+台盼蓝(TB)。然后将这两种不同
[Abstract]:Transgenic animals have a wide range of research and application prospects in medicine, life science, biopharmaceutical and other fields, so scientists have been exploring a more simple, practical and efficient transgenic method. In recent years, with the development of science and technology, gene transfer in vivo has become a very popular technology in gene therapy, biological analysis and other fields, one of which is in vivo electroporation. Spermatogonial stem cells are the progenitor of spermatozoa and the only cell type in male animals that can self-proliferate, update and transmit genetic information to the next generation. The operation and modification of spermatogonial stem cells are of great significance in the establishment of transgenic animal models and the preparation of mammary gland bioreactor. In order to explore the feasibility of injecting exogenous gene into testicular seminiferous tubules in vivo and electroporation in vivo to establish transgenic animals, We first designed in vivo electroporation of the testis of SPF grade km rats with two different parameters of electroporation (low voltage 30 ~ 110V / cm) and high voltage electric perforation (500V / cm) to observe the effects of different electroporation conditions on the degree of testicular injury and reproductive ability of SPF km male rats. In order to screen the appropriate parameters of electroporation in vivo. On the basis of this, we treated the eukaryotic expression plasmid pCE-29DNA with enhanced green fluorescent protein (EGFP) gene by two different treatments: 1) pCE-29DNA transfection reagent in vivo jetPII (TMTM) Trypan blue TBX 2) pCE-29DNA Trypan blue TBN. And then put the two different
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R-332

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