HUMAN EMBRYONIC STEM CELLS STABLY OVER-EXPRESSING EGFP MAINTAIN PLURIPOTENCY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。制备带有视觉标记的基因标记的人类胚胎干细胞。具有易于评估的遗传标记(如绿色荧光蛋白)的人类ES细胞的可用性将为许多实验机会提供便利。我们用两个EF-1α启动子替换了pCDNA3.1(Invitgen，CarlsbadCA)中的CMV和SV40启动子，制备了含有哺乳动物启动子的YPL2载体，用于在人ES细胞中进行转基因表达。在第一个EF-1α启动子的控制下插入EGFP基因，构建了YPL2-EGFP载体。第二个EF1α启动子位于新霉素抗性基因的上游。用Fugene 6将YPL2-EGFP基因导入H1或H9人胚胎干细胞，经100 ug/ml新霉素筛选12天后，观察到稳定表达EGFP的单个集落。在新霉素筛选下传代3个月后，细胞继续保持典型的HES细胞形态。免疫染色显示OCT-3/4表达维持，与未转染的HES细胞无明显区别。向细胞中加入10 ng/mlBMP4可诱导其向滋养层细胞分化，hCG分泌显著增加，细胞形态发生明显变化，但未见EGFP表达减弱。细胞传代3个月后，经流式细胞仪检测，EGFP表达无明显变化。在将EGFP-HES细胞注射到SCID小鼠体内后，有强大的畸胎瘤形成，显示出广泛的形态多能性。在这些畸胎瘤的分化细胞中，EGFP表达广泛。与慢病毒载体和IRES元件相比，该质粒用于筛选稳定的转染体的结果更加一致。携带EGFP(或其他容易识别的视觉或代谢标记)的HES细胞将在许多其他领域的研究中有用，如分化、迁移和形态发生。这项研究使用了WNPRC动物服务和研究服务，包括联邦批准的人类ES细胞系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To prepare genetically marked human embryonic stem cells with a visual marker. The availability of human ES cells with a readily evaluated genetic marker such as green fluorescent protein will facilitate a number of experimental opportunities. We replaced the CMV and SV40 promoters in pCDNA3.1 (Invitrogen, Carlsbad, CA) with 2 EF-1alpha promoters to prepare the YPL2 vector with mammalian promoters for transgene expression in human ES cells. EGFP cDNA was inserted under the control of the first EF-1 alpha promoter to construct plasmid YPL2-EGFP. The second EF1 alpha promoter was upstream of the neomycin resistance gene. H1 or H9 human embryonic stem (HES) cells were transfected with YPL2-EGFP using Fugene 6. Following 100 ug/ml neomycin selection for 12 days, individual colonies demonstrating stable EGFP expression were observed. After 3 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Immunostaining demonstrated maintenance of Oct-3/4 expression which was indistinguishable from untransfected HES cells. Adding 10 ng/ml BMP4 to the cells provoked differentiation to trophoblasts, with dramatically increased hCG secretion and morphological changes but no loss of EGFP expression. After 3 months of passage the cells showed no change in EGFP expression as determined by FACS analysis. Following injection of EGFP-HES cells into scid mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency. EGFP expression was widespread in differentiated cells of these teratomas. The results obtained with this plasmid were more consistent than with lentiviral vectors and IRES elements to select for stable transfectants. HES cells carrying EGFP (or other readily identified visual or metabolic markers) will be useful in many other areas of research such as differentiation, migration and morphogenesis. This research used WNPRC Animal Services and Research Services, including federally approved human ES cell lines.