Molecular Analysis of Embryonic Stem Cells

描述（由申请人提供）：我们对多能性和分化的分子基础知识仍然有限。这主要是由于在模型系统中开发的分子和遗传工具在人类胚胎干细胞（hESCs）中大多缺乏。为了克服这一缺点，我建议直接在hESCs中使用转座子介导的遗传筛选来识别突变体并分离控制人类细胞分化能力的基因。为了进行筛选，我们将使用遗传标记的hESC系RUES2（最初在我的实验室和NIH登记处获得并表征），它具有两种类型插入的单个副本：(i)稳定的慢病毒插入，其中人类Oct4启动子驱动EGFP和新霉素（Neo）的表达，（ii）配备有转录放大盒和湿霉素（Hyg）的诱变转座元件。我们之前已经证明这种转座因子可以被动员起来跳跃并随机插入到人类基因组中。我们建议利用这一特性来产生突变并进行无偏正向遗传筛选，如模式生物中所述。使用一种非常简单的筛选策略，基于阳性（EGFP）和阴性选择（Neo和Hyg）， 107个hESCs将被诱变并挑战其保留多能性和失去分化能力的能力。转座子的特性允许用一种直接的质粒拯救策略来表征突变基因。此外，它可以从人类基因组中切除，而不会留下痕迹来产生复合体。这些，除了通过独立手段对突变体进行表型分析外，将证实基因与突变体表型之间的联系。这种大规模的基因筛选将导致hESCs中控制细胞命运决定的基因的分离。由于hESCs为早期人类胚胎发生提供了一个窗口，这一知识将使我们更接近于理解我们自己的发育。更重要的是，这个项目的成功完成将为在人类细胞中更系统地使用无偏见基因筛选打开大门。反过来，这将对基础研究和临床应用产生重大影响。

DESCRIPTION (provided by applicant): Our knowledge on the molecular basis of pluripotency and differentiation remains limited. This is mostly due to the fact that molecular and genetic tools developed in model systems are mostly lacking in human embryonic stem cells (hESCs). To overcome this shortcoming, I propose to use a transposon mediated genetic screen directly in hESCs to identify mutants and isolate genes that control the ability of human cells to differentiate. To perform our screen we will use a genetically marked hESC line, RUES2 (originally derived and characterized in my laboratory and part of the NIH registry), that has a single copy of two types of insertions: (i) a stable lentiviral insertion where the human Oct4 promoter drives the expression of EGFP and Neomycin (Neo), (ii) a mutagenic transposable element equipped with a transcriptional amplifier cassette and Hygromycin (Hyg). We have previously shown this transposable element can be mobilized to hop and randomly insert into the human genome. We propose to take advantage of this property to generate mutations and perform an unbiased forward genetic screen, as described in model organisms. Using a very simple screening strategy, based on positive (EGFP) and negative selection (Neo and Hyg), 107 hESCs will be mutagenized and challenged for their ability to retain pluripotency and lose their ability to differentiate. The transposon has properties that allow the characterization of th mutated gene with a straightforward plasmid rescue strategy. Additionally, it can be excised from the human genome without leaving a footprint to generate revertants. Those, in addition to phenocopies of the mutant by independent means, will confirm the link between the gene and the mutant phenotype. This large-scale genetic screen will lead to the isolation of genes that control a cell fate decision in hESCs. As hESCs provide a window to early human embryogenesis, this knowledge will move us a step closer to the understanding of our own development. More importantly, the successful accomplishment of this project will open the door to a more systematic use of unbiased genetic screens in human cells. In turn, this will have a strong impact on both basic research and clinical applications.