Identification of Novel Regulators of Entry into the Meiotic Program

进入减数分裂程序的新型调节因子的鉴定

• 批准号: 240733827
• 负责人: Dr. Michelle Stevense
• 金额: --
• 依托单位: Institut für Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/240733827?language=en
• 关键词: Identification; Novel; Regulators; Entry; into

How do somatic, mitotically dividing early germ cells enter meiosis and become haploid germ cells? How do cells switch from mitotic division to meiotic division, and how are meiotic cell divisions prevented in mitotically dividing cells? Only very few factors that control the fundamental switch from mitosis to meiosis are known, despite the obvious importance of this switch for all higher eukaryotic life. The generation of gametes and functional reproductive tissue depends on this switch. The lack of knowledge in this area is in part due to difficulty in identifying cells that have just entered the meiotic program. This pilot project aims at identifying hitherto unknown factors that prevent irregular switching from mitotic into meiotic cell divisions. The removal of this factor will be the driving force that allows cells to enter meiosis in a timely manner during development and self-renewal. We have performed an esiRNA high throughput screen (HTS) using our recently generated embryonic stem (ES) cell reporter line, which specifically indicates the onset of the meiotic program by GFP expression. Eliminating factors that prevent entry into meiosis turn these cells GFP-positive, allowing us to identify such negative regulators of entry into the meiotic program. After performing a high-throughput primary screen and a secondary validation screen using an esiRNA library representing >13.000 genes, we have identified 279 candidate genes. In the one year pilot project requested to be funded, these genes will be investigated by a variety of methods including bioinformatics, knowledge-based assessment, RT-PCR expression studies in pre-meiotic and meiotic cells, and the most promising hits will be further analyzed by using or raising antibodies for protein expression, protein interaction and protein localization studies. In the mid- and long-term, after successful completion of the pilot project, we aim at fully characterizing the most promising top hits molecularly and in mouse models and expect to thereby contribute significant new insights into our understanding of the mitosis-to-meiosis transition. These discoveries will allow a deeper understanding of germ cell disease and abnormalities in the murine model, and may also have further impact in the clinical setting by future extension of the analysis to human patients.

体细胞有丝分裂的早期生殖细胞如何进入减数分裂并成为单倍体生殖细胞？细胞如何从有丝分裂分裂切换到减数分裂分裂，以及在有丝分裂分裂的细胞中减数分裂细胞如何被阻止？只有很少的因素控制从有丝分裂到减数分裂的基本转换是已知的，尽管这种转换对所有高等真核生物都很重要。配子和功能性生殖组织的产生取决于这个开关。这方面缺乏知识的部分原因是难以识别刚刚进入减数分裂程序的细胞。这个试点项目旨在确定迄今为止未知的因素，阻止从有丝分裂到减数分裂细胞分裂的不规则转换。该因子的去除将是细胞在发育和自我更新过程中及时进入减数分裂的驱动力。我们使用我们最近生成的胚胎干（ES）细胞报告系进行了esiRNA高通量筛选（HTS），该报告系通过GFP表达特异性地指示减数分裂程序的开始。消除阻止进入减数分裂的因素使这些细胞gfp呈阳性，使我们能够识别进入减数分裂程序的这种负调节因子。在进行了高通量的初级筛选和二级验证筛选后，我们使用了一个代表bb1013000个基因的esiRNA文库，确定了279个候选基因。在申请资助的为期一年的试点项目中，这些基因将通过生物信息学、知识评估、RT-PCR在减数分裂前和减数分裂细胞中的表达研究等多种方法进行研究，并通过使用或培养抗体进一步分析最有希望的hit，进行蛋白质表达、蛋白质相互作用和蛋白质定位研究。从中期和长期来看，在成功完成试点项目后，我们的目标是在分子和小鼠模型中充分表征最有希望的顶级hit，并期望从而为我们对有丝分裂到减数分裂转变的理解提供重要的新见解。这些发现将使我们更深入地了解小鼠模型中的生殖细胞疾病和异常，并可能通过将分析扩展到人类患者，在临床环境中产生进一步的影响。