小鼠胎脑组织活体转基因技术的建立

Gene Transferring into Mouse Fetal Brain In Vivo

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.