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

开发基于 lacO/lacI 的荧光报告操纵子系统来研究小鼠染色体动力学

基本信息

批准号：
10391570
负责人：
Roberto Jose Pezza
金额：
$ 21.92万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-12 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10391570
关键词：
3-Dimensional Affect Aneuploidy Articular Range of Motion Binding Biological Process Cell Nucleus Cell division Cells Centromere Characteristics Chromatin Chromosome Pairing Chromosome Segregation Chromosomes Communities Complement Congenital Abnormality Custom Data Development Disease Double Strand Break Repair Down Syndrome Engineering Environment Evaluation Fertility Fluorescence Frequencies Gene Expression Genetic Genetic Crossing Over Genetic Recombination Genome Genome engineering Genomic Segment Genomics Germ Cells Homologous Gene Hour Image Infertility Klinefelter&apos s Syndrome Lead Location MSH4 gene Mediating Meiosis Meiotic Prophase I Meiotic Recombination Methods Modeling Molecular Monitor Motion Movement Mus Mutation Phenotype Physiological Play Positioning Attribute Process Prophase Proteins Regulation Reporter Reproductive Biology Resolution Resources Role Seminiferous tubule structure Site Spermatocytes Spontaneous abortion Sterility Structure Synaptonemal Complex System Technology Testing Time Tissues Transgenic Mice Turner&apos s Syndrome Visualization Work Y Chromosome advanced system arm base chromosomal location chromosome movement cost experimental study genomic locus homologous recombination in silico in vivo innovation insight movie new technology prevent programs recruit segregation simulation telomere tool

项目摘要

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.

概括减数分裂染色体经历一系列运动，促进高度调控的染色体相互作用。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，即后来通过称为联会复合体（SC）的蛋白质结构稳定下来，该结构在同源染色体（突触）。染色体配对和 SC 形成受快速前期的影响运动（RPM）。影响染色体运动或 SC 动力学的突变可能导致代价高昂的表型从生殖生物学和生育力问题到严重的非整倍体出生缺陷。基本的有关 RPM 潜在过程的问题，以及 RPM 如何产生稳定的同源染色体配对知之甚少。同样重要的是，双链断裂对于减数分裂重组至关重要发生允许同源相互作用。然而，双链的时间和基因组分布如何？控制的了解甚少。这种控制的一个突出候选者是最近确定的 SPO11 合作伙伴， ANKRD31 蛋白，其在生殖细胞中的精确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，对小鼠的研究受到限制。该提案构建开发一种创新方法，将蛋白质引导至小鼠性母细胞的染色体位点 Vivo 利用 lacO-lacR 技术的荧光报告操作系统 (FROS)。我们的初步数据表明，该系统有潜力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 介导减数分裂双链断裂的正确时间和位置的作用机制？第一个目标将评估 RPM 对同源染色体配对的机制和调节。我们将使用 lacO/lacR-GFP 通过 3D 延时电影对哺乳动物染色体运动进行可视化和量化活的生殖细胞。我们将测试以前无法识别的染色体特征（例如染色体位置）细胞核内和染色体表达水平）调节 RPM。此外，我们新开发的长期生精管培养系统将使我们能够直接测试两个竞争模型，解释如何同源染色体相互作用并配对。这一目标将为人们对支配的动态力量提供新的见解。空间和时间上的同源配对。目标 2 将确定减数分裂中 ANKRD31 蛋白的需求双链形成。为此，我们计划生成携带 lacO 重复序列和 ANKRD31- 的转基因小鼠 GFP-lacR。将 ANKRD31 融合靶向特定基因组位点将允许直接评估 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和双链断裂形成的频率。