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鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立

发表时间:2008-06-15  浏览量:2444  下载量:706
全部作者: 俞华莉,巨兴达,王俊杰,高倩倩,伏秀青,王兴智,朱筱娟
作者单位: 东北师范大学遗传与细胞研究所,分子表观遗传学教育部重点实验室
摘 要: 体内电穿孔法转基因技术作为一种新型有效的转基因方法,被广泛应用于发育生物学,特别是中枢神经系统发育的研究。本研究旨在通过子宫内电穿孔法,建立鼠胚大脑皮层体内基因转移体系:将带有绿色荧光蛋白(GFP)的表达载体pCAGGS-IRES-EGFP显微注入胚胎期14.5 d的鼠胚侧脑室中,电极沿与脑中缝平行的方向夹持鼠脑,在电压30 V,脉冲时间50 ms,脉冲数5的条件下方波电击,将外源基因转移到室管膜层细胞。胚胎被重新放回母体内,继续发育3 d.分离被转染的鼠胚脑组织,沿冠状面冰冻切片,在荧光显微镜下观察外源绿色荧光蛋白在鼠胚大脑皮层中的瞬时表达。鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立将为进一步研究皮层发育、神经元迁移、轴突导向等以及神经发生过程中相关基因功能奠定基础。
关 键 词: 发育神经生物学;子宫内电穿孔法;显微注射;大脑皮层;鼠胚
Title: The establishment of genetic transformation system in cerebral cortex of mouse embryo via in utero electroporation
Author: YU Huali, JU Xingda, WANG Junjie, GAO Qianqian, FU Xiuqing, WANG Xingzhi, ZHU Xiaojuan
Organization: Institute of Genetics and Cytology, Northeast Normal University, Key Laboratory of Molecular Epigenetics, Ministry of Education
Abstract: Recently, as a new and effective gene transfer method,electroporation has been applied to the study of developmental biology, especially to central nervous system development. In this study, in utero transfection plasmid DNA method was set up to embryonic mouse. The GFP expression vector pCAGGS-IRES-EGFP was microinjected into the lateral cerebral ventricle of E 14.5d mouse embryos through the uterine wall. Then, the 30 V square-wave pulse was delivered across the head for 5 times and interval 50m sec by electrodes in parallel with the median raphes. Embryos were then allowed to develop for three days. The slices of transfected brain were checked under a fluorescence dissection microscope. The results showed that GFP was expressed in the lateral cerebral cortex. This system is of great significance to the further studies of cerebral cortex development, neuronal migration, axonal guidance and gain-of-function and loss-of-function induction.
Key words: developmental neurobiology; in utero electroporation; microinjection; cerebral cortex; mouse embryo
发表期数: 2008年10月第11期
引用格式: 俞华莉,巨兴达,王俊杰,等. 鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立[J]. 中国科技论文在线精品论文,2008,1(11):1300-1304.
 
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