小鼠子宫内电转基因方法的建立

Construction of in utero electroporation in the mouse

目的：建立小鼠子宫内电转基因技术，比较分析转染绿色荧光蛋白（GFP）后对胚胎发育及相关蛋白表达的影响。方法：将怀孕15d的小鼠，水合氯醛麻醉后，取出两侧子宫，用毛细管注射针将2μg／μl的pCAGGS-GFP质粒0．5-1μl准确注射到胎鼠侧脑室，在电压40V、每次脉冲60ms，间隔940ms，电脉冲6次的条件下进行定时定位活体电转基因，电转后24h取材，甲醛固定冷冻冠状切片，DAPI染细胞核观察组织形态结构变化，荧光免疫组织化学检测α-SMA的表达差异。结果：妊娠15d孕鼠转染24h后小鼠成活率80％（8／10），胚胎成活率为54．2％（13／24），存活胚胎GFP阳性表达率为61．5％（8／13），GFP阳性表达胚胎脑组织切片，基因转染区域和正常组织区组织形态结构和α-SMA表达不存在差别。结论：成功建立了小鼠子宫内电转基因的方法。

Objective： To construct the method of mouse in utero electroporation, and compare the impact of gene green fluo reseent protein （GFP） on embryonic development and protein expression after transfection. Methods： Pregnancy 15 d mice were selected, and the abdominal cavity was opened after chloral hydrate anesthesia. After 0. 5-1μl plasmid of pCAGGS-GFP was injected into the lateral ventricle of the embryonic brain, 2 uteri were electroporated under the condition of volt 40 V, pulse 60 ms, pause 940 ms for six times. In 24 h after electroporation, mouse embryos were collected and fixed with formalde- hyde, finally cut into frozen coronal slice, DAPI was used to stain nuclei showing morphology changes, and fluorescence im munoassay was carried out with the detection of α-SMA expression differences. Results： 10 mice samples were checked after transfection for 24 h with in utero electroporation, after the mice survival rate was 80% （8/10）, embryo survival rate was 54. 2 % （13/24）, survival embryonic GFP-positive expression rate was 61.5% （8/13）, and the embryonic brains with GFP-positive ex- pression were sliced for further analysis. Compared the GFP positive and normal regions in the brain, the morphology, organization and ccSMA expression did not show changes. Conclusion： The method of the in utero electroporation was constructed successfully.