Development of a lacO/lacI based flourescence reporter-operator system to study chromosome dynamics in mice

摘要 减数分裂染色体经历了一系列促进高度调控的染色体相互作用的运动。这些 相互作用最终导致母本和父本同源染色体的成对关联，这是 后来通过称为联会复合体(SC)的蛋白质结构稳定下来，这种复合体形成于 同源染色体(联会)。快速前期对染色体配对和SC形成的影响 运动(RPM)。影响染色体运动或SC动态的突变可能导致代价高昂的表型 从生殖生物学和生育问题到严重的非整倍体出生缺陷。基本原理 关于RPM的潜在过程以及RPM如何导致稳定的同源染色体的问题 人们对配对知之甚少。同样重要的是，双链断裂对于减数分裂重组是必不可少的 允许同源相互作用的发生。然而，双链的时机和基因组分布如何 人们对受控的理解很少。这种控制的一个重要候选者是最近确定的SPO11合作伙伴， ANKRD31蛋白，其在生殖细胞中的确切作用机制和靶点尚不清楚。分子 传统上，由于缺乏基因组工程工具，对小鼠的研究一直受到限制。这项建议建立了 关于发展一种创新的方法，将蛋白质定向到小鼠卵母细胞的染色体上 利用使用LACO-LACR技术的荧光报告-操作者系统(FROS)。我们的预赛 数据显示，这一系统有可能回答以前难以解决的问题 小鼠减数分裂与雄性生殖细胞的分化和成熟有关。计划中的实验将 回答两个具体问题：1)染色体运动如何促进稳定的同源配对；2)什么是 ANKRD31在调节减数分裂双链断裂的适当时机和位置中的作用机制？ 第一个目的将评估RPM对同源染色体配对的机制和调节。我们会 使用LACO/LACR-GFP在哺乳动物中使用3D时间推移电影可视化和量化染色体运动 活的生殖细胞。我们将测试以前未识别的染色体特征(例如，染色体定位 在核和染色体水平的表达)，调节RPM。此外，我们新开发的 长期的生精小管培养系统将允许我们直接测试两个相互竞争的模型，解释如何 同源染色体相互作用和配对。这一目标将使人们对统治的动态力量有新的见解。 空间和时间上的同源配对。目标2将确定ANKRD31蛋白在减数分裂中的需求 形成双链。为此，我们计划培育携带LACO重复序列和ANKRD31的转基因小鼠。 GFP-LACR。将ANKRD31融合到特定的基因组座位将允许直接评估ANKRD31对 SPO11辅助蛋白(REC114、MEI4和IHO1)的局部积累，下游重组热点 中间体，以及双链断裂形成的频率。

SUMMARY Meiotic chromosomes undergo a range of motions promoting highly regulated chromosome interactions. These interactions culminate in pairwise associations of maternal and paternal homologous chromosomes, which are later stabilized via the proteinaceous structure called synaptonemal complex (SC) that forms between the homologous chromosomes (synapsis). Chromosome pairing and SC formation are influenced by rapid prophase movements (RPMs). Mutations that affect chromosome motions or SC dynamics can lead to costly phenotypes ranging from problems in reproductive biology and fertility to severe aneuploid-based birth defects. Fundamental questions regarding the processes underlying RPMs, and how RPMs lead to stably homologous chromosome pairing are poorly understood. Equally important, double-strand breaks are essential for meiotic recombination to occur allowing homologous interactions. However, how timing and genome distribution of double strand are controlled is poorly understood. A prominent candidate for this control is the recently identified SPO11 partner, the ANKRD31 protein, whose precise mechanism of action and targets in germ cells are unknown. Molecular studies in mouse have been traditionally limited by the lack of genome engineering tools. This proposal builds on the development of an innovative approach that directs proteins to chromosomal loci in mouse meiocytes in vivo by utilizing a fluorescence reporter-operator system (FROS) using lacO-lacR technology. Our preliminary data shows that this system has the potential to answer questions that have previously been intractable for mouse meiosis relevant to differentiation and maturation of male germ cells. The planned experiments will answer two specific questions: 1) how do chromosome motions promote stable homolog pairing and 2) what is the action mechanism of ANKRD31 in mediating proper timing and location of meiotic double strand breaks? The first Aim will assess the mechanism and regulation of RPMs on homologous chromosome pairing. We will use lacO/lacR-GFP to visualize and quantify chromosome motions using 3D time-lapse movies in mammalian live germ cells. We will test how previously unrecognized chromosome characteristics (e.g. chromosome location within the nucleus and chromosomal level of expression) that modulate RPMs. Additionally, our newly developed long-term seminiferous tubule culture system will allow us to directly test two competing models explaining how homologous chromosomes interact and pair. This aim will lend new insights into the dynamic forces that govern homolog pairing in space and time. Aim 2 will determine the requirements of the ANKRD31 protein in meiotic double strand formation. To this end we plan to generate transgenic mice carrying lacO repeats and ANKRD31- GFP-lacR. Targeting ANKRD31 fusion to specific genomic loci will allow direct evaluation of ANKRD31 effect on local accumulation of SPO11 auxiliary proteins (REC114, MEI4, and IHO1), downstream recombination hotspot intermediates, and frequency of double strand break formation.