权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

总结