Chromatin Expulsion by the DNA Replication Licensing Factor ORC4 during Asymmetric Cell Division in Meiosis and Differentiation

在这个修订的R01申请中描述的研究目的是探索DNA许可因子ORC4在雌性减数分裂期间通过笼形形成将染色体隔离到极体中的作用。这些研究被认为是极具创新性的，对理解雌性减数分裂和卵母细胞成熟的机制以及其他可能的不对称细胞分裂形式具有重要意义。该申请的其他优势包括首席研究员的杰出资格和生产力记录，招募了一个具有互补专业知识的强大合作团队，夏威夷大学非常有利的研究环境，以前出版物形式的令人信服的前提和初步数据确认ORC4的潜在功能。实现项目目标所需的许多关键试剂和资源的可用性，对科学严谨性要素的充分关注以及对先前批评的强烈回应。除了这些丰富的优势之外，一些人对不发达的方法表示担忧，包括缺乏对涉及ORC4消耗的实验的机制影响的指导，超出先前的观察，担心EGFP-ORC-4融合蛋白的过度表达可能诱导细胞应激或致死，ORC4相互作用伙伴的纯化缺乏可行性，以及对ORC4在其他形式的不对称细胞分裂中的作用缺乏足够的了解。虽然这些和其他一些小的缺点略微降低了应用程序的优点，但专家组最终得出结论，认为将获得的非常高的新颖性和基本机制见解将对卵母细胞发育和发育生物学领域产生重大影响。

The goal of studies described in this amended R01 application is to explore the role of the DNA licensing factor, ORC4, in sequestering chromosomes into polar bodies via cage formation during female meiosis. These studies were considered highly innovative and with fundamental significance for understanding the mechanism of female meiotic division and oocyte maturation, as well as other possible forms of asymmetric cell division. Other strengths of the application include the outstanding qualifications and record of productivity of the Principal Investigator, the recruitment of a strong collaborative team with complementary expertise, the highly conducive research environment at the University of Hawaii, the convincing premise in the form of previous publications and preliminary data confirming the underlying functionalities of ORC4, the availability of many key reagents and resources required to pursue the project goals, adequate attention to elements of scientific rigor and a strong response to the previous critiques. In addition to these abundant strengths, some expressed concerns with an underdeveloped approach, including lack of guidance as to the mechanistic implications for experiments involving ORC4 depletion, beyond previous observations, concerns that overexpression of the EGFP-ORC-4 fusion protein may induce cell stress or lethality, lack of feasibility of purification of ORC4 interacting partners and the absence of sufficient insight on the role of ORC4 in other forms of asymmetric cell division. While these and other minor weaknesses slightly reduced the perceived merit of the application, the panel ultimately concluded that the very high novelty and fundamental mechanistic insights to be gained will exert a major impact on the fields of oocyte development and developmental biology. rtility.