HUMAN EMBRYONIC STEM CELLS STABLY OVER-EXPRESSING EGFP MAINTAIN PLURIPOTENCY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。制备具有可视标记的遗传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ES细胞的可用性将促进许多实验机会。我们用2个EF-1 α启动子替换pCDNA3.1（Invitrogen，卡尔斯巴德，CA）中的CMV和SV 40启动子，以制备具有哺乳动物启动子的YPL 2载体，用于在人ES细胞中转基因表达。将EGFP cDNA插入第一个EF-1 α启动子的控制下，构建质粒YPL 2-EGFP。第二个EF 1 α启动子位于新霉素抗性基因的上游。使用Fugene 6用YPL 2-EGFP转染H1或H9人胚胎干（HES）细胞。在100 μ g/ml新霉素选择12天后，观察到显示稳定EGFP表达的单个集落。在新霉素选择下传代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表达的维持，其与未转染的HES细胞难以区分。向细胞中加入10 ng/ml的BMP 4可诱导细胞分化为滋养层细胞，hCG分泌和形态学变化显著增加，但EGFP表达没有损失。在传代3个月后，通过FACS分析测定，细胞显示EGFP表达没有变化。在将EGFP-HES细胞注射到scid小鼠中后，存在稳健的畸胎瘤形成，其证明了广泛的形态学多能性。EGFP表达广泛存在于这些畸胎瘤的分化细胞中。用该质粒获得的结果比用慢病毒载体和IRES元件选择稳定的转染子更一致。携带EGFP（或其他容易识别的视觉或代谢标记物）的HES细胞将在许多其他研究领域（如分化、迁移和形态发生）中有用。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.