小鼠胎脑组织活体转基因技术的建立  

Gene Transferring into Mouse Fetal Brain In Vivo

作　　者：吴燕[1] 丁学锋[1] 刘淑红[1] 赵永岐[1] 吴彦瑞[1] 王飞[1] 范文红[1] 范明[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2010年第3期413-415,共3页Letters in Biotechnology

基　　金：国家自然科学基金(30871030);国家重点基础研究发展计划(2006CB504100)

摘　　要：目的:建立实用的小鼠活体脑组织基因转染技术。方法:将EGFP质粒(CAGS启动子)注射到胎龄16 d(E16d)的胎鼠侧脑室,用镊形电极隔着子宫壁夹住胎鼠头部,在45 V电压下给予5次电脉冲刺激,每次刺激50ms,间隔1 s;转染后不同天数将胎鼠脑组织完整取出,以4%PFA固定后冰冻切片,进行激光共聚焦照相。结果:EGFP质粒被转入小鼠活体脑组织细胞中并获得表达,动物存活率为90%,GFP阳性率高于80%。结论:通过对麻醉剂、电脉冲刺激、质粒浓度、术中术后处理等多种实验条件的摸索,建立了实用的小鼠胎脑组织活体转基因技术。Objective：To establish a method of transferring gene into mouse fetal brain in vivo.Methods：EGFP（CAGS Promoter） plasmids were injected into the lateral ventricle of embryonic day 16（E16d） using the mouth-controlled micropipette under the illumination of a fiber optic light source.Hold the DNA-injected embryonic head through the uterus with forceps-type electrodes and deliver five electric pulses with an electroporator.The voltage is 45 V and electric pulses are delivered every other second,with a duration of 50 ms per pulse.The embryonic brain were fixed by 4% PFA and made into frozen sections.Scan the migrating neurons with confocal microscope.Results：The EGFP plasmid were successfully transferred into neurocytes in vivo,more than 90% of operated em-bryos survive and more than 80% of these survivors express GFP appropriately.Conclusion：By exploring the con-dition of anesthetic agent,electric impulse,plasmid concentration and treatments in the operation,we successfully established the method of transferring exogenous gene into mouse fetal brain in vivo.

关 键 词：子宫内电转 转基因 神经元迁移 胚胎 

分 类 号：Q78[生物学—分子生物学]