【作者】 冯蕾；

【导师】 韩骅；

【作者基本信息】 第四军医大学 ， 发育生物学， 2008， 博士

【摘要】 转基因小鼠是最早建立的转基因动物。此项技术是近年来生命科学中最热门,发展最快的领域之一,已经成为现代生物学家不可缺少的研究工具。它可以通过对基因的操作,直接观察基因在活体内的活动情况及其表达产物所引起的表型效应。其已广泛应用于分子生物学、免疫学、发育生物学、制药及畜牧育种等各个研究领域中,既具有深远的理论价值,又有重大的应用价值。成功高效的基因转移是转基因动物制备的关键,依据基因转移方式的不同目前制作转基因小鼠的方法有十多种之多。受精卵原核显微注射法是当前最常用最有效的建立转基因动物的方法之一。其基本过程是利用显微操作系统和显微注射技术,用直径约为1μm的玻璃管直接插入受精卵的雄原核内,将毛细管内所带有的外源基因的DNA注入受精卵原核,使外源基因整合到动物基因组,再通过胚胎移植技术将整合有外源基因的受精卵移植到受体的输卵管或子宫内继续发育,产下的携带有外源基因的子代即得到转基因动物。受精卵原核显微注射法是目前基因转移效率较好的一种基因转移方法,为了实现对多种基因的快速准确的分析,我们精心建立了小鼠受精卵原核显微注射系统。包括显微注射室的改造,仪器的购置与安装,不孕雄鼠的结扎,供体小鼠的超排卵,假孕雌鼠的制作,原核注射及胚胎输卵管移植等各个环节。成功建立了制作转基因小鼠的受精卵原核显微注射系统平台。受精卵原核注射法是一项技术性很强的工作,操作精细,涉及的步骤繁杂,影响因素多,稍有不慎便得不到预期的结果。我们又在如何提高实验结果的稳定性和整合效率方面,进行了一些有益的探索和技术优化。建立了提高实验结果及稳定性的优化方案。实验证明用3~5周龄,体重12~14g的CBAXC57BL/6的杂交一代F1雌鼠,第1天13:30腹腔注射5IU PMSG,第3天12:30注射5IU hCG后立即与雄种鼠合笼,第4天10:30取卵,能得到最好的超排数量及质量的受精卵细胞。雄原核注射时每个卵细胞注射1~2μg/ml(2~4pl)的线性化的纯化的DNA溶液,受精卵成活,发育成2细胞率最高。胚胎移植时每只受体雌鼠每侧移植10~15枚,产仔率最佳。KM是经济又好用的受体鼠种。我们建立了提高实验结果及稳定性的优化方案。基于此项技术我们构建了在内皮细胞特异性表达可诱导Tie2-CreERTM转基因小鼠。首先我们构建了在内皮细胞中特异性表达Cre重组酶的可诱导的转基因表达载体Tie2-CreER TM-core enhancer和Tie2-CreER TM-full enhancer,通过显微注射方法,注射入C57BL/6与CBA的杂交F1代小鼠受精卵原核中,移植到受体小鼠的输卵管伞部,生下的仔鼠用PCR的方法扩增基因组DNA,共筛选出3系Tie2-CreER TM core enhancer和1系Tie2-CreER TM-full enhancer转基因小鼠。此转基因小鼠可作为血管内皮细胞谱系分析和在血管内皮细胞进行条件基因打靶的理想工具小鼠。如用此转基因小鼠与我室拥有的转基因RBP-Jfloxed小鼠交配后,仔鼠注射它莫西酚即可诱导在血管内皮细胞条件性剔除RBP-J基因。通过组织学及免疫组织化学染色等即可观察血管内皮细胞在Notch/RBP-J信号阻断后的血管生成及表达标记的变化。更多还原

【Abstract】 Transgenic mice were the earliest transgenic animals. This technology is developing fast in the life science for the past few years. It is already be the necessary research tool for the modern biologist. We can direct observing expressive product of the gene and the phenotypic effect though manipulate with the gene in vivo. This technology is used widespread in molecular biology, immunology, developmental biology and livestock breeding. It has a very important role in the life science research.The key point in making a transgenic animal is transferring gene successfully into the mouse eggs. There are more than ten methods to make transgenic mouse according to the different gene transfer method. A method for microinjection of mouse zygotes to produce transgenic mice is the commonly used and effectively transfered method in present. Its elementary process is to push the injection pipette which with 1μm diameter into the pronucleus of eggs -7- and inject the exogenous gene DNA solution into it. Then transfer the injected eggs into the oviduct of acceptor female mouse. The pups were born which take along the exogenous gene are the transgenic mice.This method is the effective way to make transgenic mice. To analyze gene in vivo quickly and precisely, we have set up a mouse fertilized zygote microinjection system. Including set up the lab equipment, vasectomizing male mouse, inducing superovulation, making pseudopregnancy female mouse, microinjecting and oviduct transplanting.In addition, injection into the mouse pronucleus of fertilized zygote is the classic transgenic technology. It is still the main method to transgenic animal till now. But this technology has many steps and requires operation with highly attention. To improve stability of gene expression and integration efficiency we research and optimizate many aspects of the technology.We found that IP 3~5 weeks old, body weight 12~14g CBAXC57BL/6 F1 hybrid mouse 5 IU PMSG at 13:30 in the first day, IP5IU hCG at 12:30 in the third day, then mated with stud male mouse immediately. We get the eggs at 10:30 in the fourth day. The quantity and qualitative of the eggs were the best ones. The fertilized eggs have developmented into 2 cell stage rate is the highest when we inject 1~2μg/ml(2~4pl)linearized purified DNA solution into the male pronucleus. The number of the pups were maximum when we transferred 10~15 injected eggs into the oviduct of receptor female mouse. KM is the good receptor female mouse.We generated vascular endothelial cell specific expression of inducible Tie2-CreERTM transgenic mice. First we constructed the expression vector Tie2-CreERTM-core enhancer and Tie2-CreERTM-full enhancer. We have microinjected the linearized purified DNA solution into fertilized C57BL/6 XCBA F1 oocytes. The genotypes of all offsprings were analyzed by PCR on genomic DNA from tail biopsies. Total three founder mice carrying the Tie2-CreERTM-core enhancer and one founder carrying Tie2-CreERTM-full enhancer. The integration efficiency is 6%and 5%.The Tie2-CreERTM mouse we generated in current study is a valuable genetic tool not only for endothelial cell-specific gene targeting, but also useful for endothelial cell lineage analyses. Our lab generated the conditional knock-out mouse model of the key transcription factor RBP-J of Notch signaling RBP-J flox/flox. This model mated with Tie2-CreERTM mouse. The pups will conditional knock-out RBP-J and then block the Notch signaling in endothelial cells after tamoxifen induction. Then can reveal the angiogenesis and changing of the makers though histology and immunohistochemistry.更多还原