绿色荧光蛋白嵌合体小鼠的建立和鉴定  

Establishment and determination of chimeric mice stably expressing green fluorescent protein

作　　者：招霞[1] 马芸[1] 陈系古[1] 葛坚[1] 黄冰[1] 邓新燕[1] 喻瓴[2] 赖英荣[3] 黄春浓[4] 

机构地区：[1]中山大学实验动物中心,广州510080 [2]中山大学眼科中心,广州510080 [3]中山大学中山医学院病理教研室,广州510080 [4]中山大学中山医学院遗传教研室,广州510080

出　　处：《动物学报》2004年第5期784-790,共7页ACTA ZOOLOGICA SINICA

基　　金：国家"九五"攻关课题 (No .10 10 3 3 );广东省科技计划重大项目基金 (No .B60 2 );广州市科技计划项目基金资助项目~~

摘　　要：为研究嵌合体动物中供体胚胎干细胞 (ES)在宿主胚胎发育中的走向和定位 ,同时探讨绿色荧光蛋白(GFP)基因作为报告基因在转基因动物制作中的应用价值 ,本研究将pEGFP N1基因导入小鼠ES D3 细胞系 ,得到稳定表达GFP的胚胎干细胞亚系ES D3 GFP ,通过对昆明小鼠的囊胚腔注射 ,获得了 4只表达绿色荧光蛋白的嵌合体小鼠。其中 1只存活至成年 ,3只出生时死亡。荧光显像及组织PCR检测显示了绿色荧光蛋白在小鼠体内的嵌合情况。以绿色荧光为指标可实现活体水平的动态观察 ,本实验首次观察到以GFP为指标所示的机体嵌合情况与根据毛色嵌合推测的机体嵌合情况存在很大差异 ,以GFP为嵌合指标更加全面而准确 ;但不排除GFP对小鼠发育存在一定毒性的可能 ;另外 ,有结果显示供体ES细胞在宿主体内除了大片补丁状嵌合外 ,还存在细胞散在嵌合的情况 ,后者提示了在组织中利用GFP对ES细胞实施单细胞追踪和实时观察的可行性 。In order to follow the fate and migration of embryonic stem (ES) cells during host mouse development, and investigate the utilization of green fluorescent protein (GFP) as a reporter gene tracing ES cells in vivo, we transfected pEGFP-N 1 gene into ES-D 3 cells and generated an ES cell line expressing GFP efficiently and stably. These ES cells were introduced into host embryos from outbred KMW though blastocyst injection. Using GFP, the attractive genetic marker, we have been able to follow the fate of ES cells in living embryos and to observe directly the contribution of these cells to mouse embryos. Totally, four GFP-expressing ‘green’ chimeric mice expressing GFP were obtained. One has developed to an adult and three died when they were born. Fluorescence imaging and PCR analysis showed the existence of GFP in the mice. For the first time, we observed that there was an obvious difference between the results of chimerism analysis by detecting the expression of GFP gene or by coat analysis. GFP marker is more accurate and comprehensive. However, it should not be excluded that the expression of GFP gene may be detrimental to mouse development. We also showed that individual EGFP expressing cells were visualized within a chimeric population. This suggests that real-time tracking of() cells can be performed by allowing multiple experimental time points to be obtained from a single sample.

关 键 词：小鼠 嵌合体 体内 观察 供体 ES细胞 宿主 绿色荧光蛋白(GFP) 绿色荧光蛋白 体胚 

分 类 号：Q819[生物学—生物工程]