运用电穿孔技术建立小鼠脑内基因转染模型  

The Method of in vivo Gene Transfection into Mouse Brain by Means of in Utero Electroporation

作　　者：杜鹏[1] 王春雷[2] 徐运兰[1] 喻志源[1] 谢敏杰[1] 王伟[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030 [2]中国科学院上海神经科学研究所,上海200030

出　　处：《华中科技大学学报（医学版）》2007年第4期504-506,559-560,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金重点资助项目(No.30230140;No.30400142);教育部新世纪优秀人才支持计划(NCET-04-0695)

摘　　要：目的借助生物物理技术建立一种高效实用的脑内基因转染技术,探讨其在神经科学研究中的应用。方法选取孕15 d C57系小鼠,取出子宫角,用自制玻璃电极将目的质粒和荧光质粒混匀后注入胎鼠侧脑室,应用BTX电转染仪在胎鼠脑两颞极间给予设定的电脉冲刺激,后将胎鼠放入孕鼠腹腔让其自然出生后8 d取脑,冰冻切片,抗GFP、抗Tubulin免疫组化染色,荧光显微镜下观察。结果在小鼠大脑皮层Ⅱ、Ⅲ层,转染CAG-EGFP的脑片可见绿色荧光细胞带,转染细胞抗GFP免疫组化染色阳性;转染DS-red的呈红色荧光细胞带,转染细胞均呈抗Tubulin免疫组化染色阳性。结论子宫内胚胎电转染技术是一个很好的在体基因转染技术,可为神经科学研究提供一个很好的技术平台。Objective To explore an efficient and applicable method of in vivo gene transfection into the mouse brain. Methods Uterine horns of 15.5-day pregnant mice were exposed, and plasmid was injected into the lateral ventricle of embryos or embryonic mice. Embryonic brains were placed between tweezers-type electrodes, subjected to special square electric pulses by an electroporator, and then quickly taken back into the abdominal cavity of pregnant mice. Embryos were allowed to develop normally and sacrificed on postnatal 8th day. Brains were fixed, cryopreserved, frozen, and 20 μm thick sections were obtained and immunostained with anti-GFP and anti-Tubulin. Results There were fluorescent cell zones in the Ⅱ , Ⅲ layer of the cerebral cortex of gene transfected brain. The slice transfected with CAG-EGFP displayed green fluorescent cell zones which were positive for anti-GFP immunostaining. The slice transfected with DS-red displayed red fluorescent cell zones. All the transfected cells were positive for anti Tubulin immunostaining. Conclusion The in utero electroporation technique is a powerful technique for the introduction of DNA into the mouse brain.

关 键 词：子宫内电转染 基因转染 小鼠 

分 类 号：Q343.1[生物学—遗传学]