Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节
Summary
我们描述记者荧光法快速识别和表征基因调控小鼠胚胎干细胞维持和自我更新。
Abstract
两个定义特征的胚胎干细胞（ES细胞）的全能性和自我更新。了解潜在的分子机制，将大大方便使用胚胎干细胞的发育生物学研究，疾病模型，药物发现和再生医学（1,2审查）。
为了加快胚胎干细胞的维持和自我更新的小说监管的识别和表征，我们开发了一种基于荧光记者分析定量测量使用Oct4GiP细胞3在小鼠胚胎干细胞的自我更新的状态。 Oct4基因启动子区域4,5的控制下，Oct4GiP细胞表达绿色荧光蛋白（GFP）。 Oct4的需要为ES细胞的自我更新，高度在ES细胞中表达，并迅速下调在分化6,7。因此，GFP的表达和记者的细胞荧光相关忠实与胚胎干细胞的身份5，荧光激活细胞分选仪（FACS）分析可以用来密切注视3,8在单细胞水平细胞的自我更新的状态。
用RNAi耦合，Oct4GiP的记者法可用于快速识别和研究的胚胎干细胞维持和自我更新3,8监管。化验自我更新的其他方法相比，它更方便，灵敏，定量，成本更低。它可进行96 – 或384孔板为大规模的研究，如高通量筛选或遗传上位性分析。最后，通过使用其他血统的特定记者ES细胞系，我们在这里描述的检测，还可以进行修改，研究胚胎干细胞分化过程中的命运规范。
Protocol
1。 Oct4GiP小鼠胚胎干细胞维持请提供由奥斯汀·史密斯博士Oct4GiP细胞。他们来自129/Ola携带Oct4的4,5转基因GFPiresPac小鼠。他们均保持在明胶涂层的组织培养皿中的ESGRO完整加克隆的高档中型（Millipore公司），或在的M15培养基：DMEM培养基（Invitrogen公司）15％胎牛血清，1000 U / ml的ESGRO（Millipore公司），1个非补充人体必需的氨基酸（Invitrogen公司），1X EmbryoMax Nucleasides（Millipor…
Discussion
我们上面描述的Oct4GiP记者分析，可以定量衡量的自我更新与分化程度。其他可用的方法，如12基于形态和增殖/活力检测，比较，它提供了更高的灵敏度和吞吐量，以及胚胎干细胞状态的一个更直接的测量。因此，非常适合大型屏幕和遗传上位性分析。事实上，我们和其他人已成功地用于全基因组RNAi屏幕3,8 Oct4GiP记者分析。然而，所有实验一样，它也有其局限性。它只能被用来研?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢阅读和编辑稿件：布拉德Lackford。这项研究是由美国国家环境健康科学，健康院内研究计划Z01ES102745国家研究院（生长激素）研究所的支持。
Materials
| Name of the reagent | Company | Catalogue number |
| ESGRO complete plus clonal grade medium | Millipore | SF001-500P |
| DMEM (High glucose 1X) | Invitrogen | 11965 |
| 0.25% Trypsin-EDTA | Invitrogen | 25200 |
| Lipofectamine 2000 | Invitrogen | 1001817 |
| OPTI-MEM(reduce serum medium) | Invitrogen | 31985 |
| ESGRO mLIF (107 units/1ml) | Millipore | DAM1776540 |
| MEM NEAA (Non-Essential Amino Acids) | Invitrogen | 11140 |
| 100x Nucleosides for ES cell | Millipore | 10620-1 |
| 2-mercaptoethanol | Sigma | M7522-100ml |
| ES-qualified fetal bovine serum | Invitrogen | 10437 |
| Nanog siRNA | Invitrogen | MSS231181 |
| Oct4 siRNA | Dharmacon | D-046256-02 |
| Sox2 siRNA | Dharmacon | M-058489-01 |
Control siRNA: siRNA duplex targeting firefly luciferase (5′-CGTACGCGGAATACTTCGA) synthesized by Dharamcon.
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Tags
Oct4GiPReporter AssayGenesRegulateMouse Embryonic Stem Cell MaintenanceSelf-renewalPluripotencyMolecular MechanismDevelopmental Biology StudiesDisease ModelingDrug DiscoveryRegenerative MedicineFluorescence Reporter-based AssaySelf-renewal StatusMouse ES CellsOct4 Gene Promoter RegionGreen Fluorescent Protein (GFP)DifferentiationFluorescence-activated Cell Sorting (FACS) AnalysisRNAiConvenienceSensitivityQuantitative AnalysisCost-effective
Citazione di questo articolo
Zheng, X., Hu, G. Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal. J. Vis. Exp. (63), e3987, doi:10.3791/3987 (2012).