DNA Replication during ES Cell Differentiation

描述(由申请人提供)： 这个K18提案的总体目标是开发一个研究小鼠(MES)和人类(HES)胚胎干细胞(ES)分化过程中DNA复制的计划。这位候选人对我们理解DNA复制在细胞周期中是如何调控的做出了重大贡献，现在他希望将这一专业知识应用于哺乳动物早期发育过程中复制的研究。在已建立和/或分化的细胞系之间已经观察到起源规格和复制时间的变化。这些变化与大范围的染色质重塑相吻合，并可能发生在主要细胞命运决定的早期发育阶段。我们将开发MES和HES细胞系统，MES细胞提供复杂的遗传工具和分化系统。一个目标是在MES细胞分化的不同阶段进行全基因组复制计时和基因表达分析，并检查特定突变对复制程序调节的影响。相比之下，我的HES细胞具有几乎完全测序的基因组的优势，允许构建尚未用于小鼠的完全覆盖的DNA阵列。由于哺乳动物的复制起始点不是由明显的DNA序列提供的，而且由于它们在很大程度上被排除在转录单位之外，因此它们的无偏见识别需要完全覆盖的基因组阵列。因此，第二个目标是与耶鲁大学基因组分析中心的迈克·斯奈德合作，确定人类22号染色体长度上复制起点的位置。我们特别感兴趣的是识别在发育过程中受到调控的起源的可能性，这将需要HES细胞的同质分化系统，与最近在小鼠中开发的细胞相平行。HES单元由SLA#03-W134，WiCell Ref P98222US以及单元线WA01和WA09引用。这一奖项将与休假同时进行，休假将从访问Geron，Co.(加利福尼亚州门洛帕克)开始，为期2-3周，学习他们最新的HES分化技术，然后在家里的实验室工作，绘制复制起源图。第二阶段将涉及2-3个月的彼得和喜悦Rathjen(阿德莱德大学)，他们已经开发了通过定义的中间体均匀分化MES细胞的系统。同质分化将使我们能够确定发生特定复制时间变化的精确细胞周期。MES细胞的同质分化条件也将应用于HES细胞，旨在生产用于复制研究的HES细胞分化中间体。

DESCRIPTION (provided by applicant): The overall goal of this K18 proposal is to develop a program for the study of DNA replication during the differentiation of mouse (mES) and human (hES) embryonic stem (ES) cells. The candidate has made significant contributions to our understanding of how DNA replication is regulated during the cell-cycle and would now like to apply this expertise to the study of replication during early mammalian development. Changes in origin specification and replication timing have been observed between established and/or differentiated cell lines. These changes coincide with large scale chromatin remodeling and likely take place early in development during major cell fate decisions. We will develop both mES and hES cell systems, mES cells offer sophisticated genetic tools and differentiation systems. One objective will be to perform genome-wide replication timing and gene expression analysis at different stages during the differentiation of mES cells, and to examine the effects of specific mutations on the regulation of the replication program. By contrast, I hES cells have the advantage of a nearly completely sequenced genome, allowing for the construction of complete coverage DNA arrays that are not yet available for mice. Since replication origins in mammals are not spec fed by obvious DNA sequences, and since they are largely excluded from transcription units, their unbiased identification requires complete-coverage genomic arrays. Hence, a second objective is to identify the positions of replication origins along the length of human chromosome 22, in collaboration with Mike Snyder at the Yale Genome Analysis center. Particularly interesting to us is the potential to identify origins that are regulated during development, which will require homogeneous differentiation systems for hES cells paralleling those recently developed in mouse. The hES cells are referenced by SLA#03-W134, WiCell Ref P98222US and as cell lines WA01 and WA09. This award would coincide with a sabbatical, which will begin with a 2-3 week visit to Geron, Co. (Menlo Park, CA) to learn their latest hES differentiation techniques, followed by work in the home Labto map replication origins. A second phase will involve a 2-3 month period with Peter and Joy Rathjen (U. of Adelaide), who have developed systems to differentiate mES cells homogeneously through defined intermediates. Homogeneous differentiation will allow us to identify the precise cellcycle during which specific replication timing changes take place. Conditions for homogeneous differentiation of mES cells will also be applied to hES cells in work aimed at the production of hES cell differentiation intermediates for replication studies.