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

• 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
• 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' 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' 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

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.

总结 减数分裂染色体经历一系列运动，促进高度调节的染色体相互作用。这些 相互作用最终导致母本和父本同源染色体的成对结合， 后来通过称为联会复合体（SC）的蛋白质结构稳定下来， 同源染色体（联会）。染色体配对和SC的形成受快速前期的影响 运动（RPM）。影响染色体运动或SC动力学的突变可导致昂贵的表型 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 关于RPM的潜在过程的问题，以及RPM如何导致稳定同源染色体 对配对的了解很少。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。然而，双链的时间和基因组分布是如何 人们对控制的了解很少。这种控制的一个突出的候选者是最近确定的SPO 11伙伴， ANKRD 31蛋白，其精确的作用机制和生殖细胞中的靶点尚不清楚。分子 在小鼠中的研究传统上受到缺乏基因组工程工具的限制。这一建议建立 关于开发一种创新的方法，将蛋白质引导到小鼠性母细胞的染色体位点， 通过使用lacO-lacR技术的荧光标记-操作系统（FROS）在体内进行。我们的初步 数据显示，该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 ANKRD 31在介导减数分裂双链断裂的正确时间和位置中的作用机制？ 第一个目标是评估RPMs对同源染色体配对的机制和调控。我们将 使用lacO/lacR-GFP在哺乳动物中使用3D延时电影来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被识别的染色体特征（例如染色体位置） 在细胞核内和染色体水平的表达），其调节RPM。此外，我们新开发的 长期曲细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。这一目标将使我们对支配全球经济的动态力量有新的认识。 空间和时间上的同源配对。目的2将确定ANKRD 31蛋白在减数分裂中的需求 双链形成。为此，我们计划产生携带lacO重复序列和ANKRD 31 - 1的转基因小鼠。 GFP-lacR。将ANKRD 31融合物靶向至特定基因组基因座将允许直接评估ANKRD 31对细胞增殖的作用。 SPO 11辅助蛋白（REC 114、MEI 4和IHO 1）的局部积累，下游重组热点 中间体和双链断裂形成的频率。