Toward a library of balanced neuronal gene knockouts in C. elegans

建立秀丽隐杆线虫平衡神经元基因敲除文库

• 批准号: 9760330
• 负责人: MATTHEW SAUL RICH
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760330
• 关键词: Adult; Affect; Aldicarb; Alleles; Animal Model; Animals; Axon; Biological Assay; Biology; Caenorhabditis elegans; Catalogs; Cells; Characteristics; Chemicals; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Data; Defect; Development; Disease; Engineering; Equilibrium; Essential Genes; Etiology; Gene Deletion; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Processes; Genetic Screening; Genetic screening method; Genome engineering; Growth Cones; Image; Knock-out; Label; Levamisole; Libraries; Maintenance; Methods; Molecular; Mutagenesis; Mutation; Nervous system structure; Neurons; Organism; Pattern; Phenotype; Process; Proteins; Reagent; Reporting; Research Personnel; Resistance; Schizophrenia; Screening procedure; Signal Transduction; Signaling Molecule; Site; Structural Protein; Structure; Synapses; TNFSF5 gene; Testing; Tissues; Work; autism spectrum disorder; cell motility; cell type; experimental study; fluorescence imaging; gene function; genetic analysis; genome-wide; interest; knockout gene; mutant; nervous system disorder; neuron development; next generation; novel; programs; protein structure; synaptic function; tool; transgene expression; transposon sequencing

PROJECT SUMMARY Dysregulation of the molecular processes that contribute to synaptic development, maturation, and stabilization underlie many neurological diseases like autism and schizophrenia. Formation and long-term maintenance of synapses requires the concerted effort of many proteins – cell motility factors guide growth cones toward post- synaptic cells, signaling molecules sense when synapses are formed, and structural proteins strengthen and stabilize the developing synapse. Analyzing the genetics of these processes is difficult, though, as many of these genes are essential or redundant. Despite decades of forward genetic screening in C. elegans and other model organisms, there are still many open questions regarding the genetics of the synapse. For instance, what signal does a growth cone migrate toward? How does a newly-formed synapse signal that it needs stabilization? How do deficiencies in these processes contribute to disease etiology? Finding the genes that will help to answer these questions using current genetic screening tools is untenable. We require next- generation genetic tools that will allow us dissect and analyze these processes. I will develop MosTrap, a novel gene-trap mutagenesis method that can be used for forward genetic screening. When inserted in a gene, the MosTrap transposon inactivates the gene and expresses both a fluorescent marker to report expression patterns and a phenotypic rescue gene that can be used to select for inactivation of genes expressed in a cell type of interest. This rescue balances the gene deletion, potentially enabling the creation of a knockout allele for all genes. Using MosTrap, I will create a collection of balanced knockout alleles in neuronal genes. During the creation of this collection, I will phenotype knockout strains for defects in synaptic function, as well as characterize which genes are essential and in what cells each gene is expressed. I will then screen the mutants in this collection for knockouts in genes that are required for synaptic stability. Using longitudinal and live imaging I will characterize the synaptic stability defects in each strain. Together, these data will identify new synaptic stability genes, as well as inform the basic biology of how synapses become unstable, potentially uncovering new mechanisms that may be utilized to treat diseases like autism. The reagents and methods I develop during this work enable the creation of a genome-wide collection of balanced knockout alleles.

项目摘要 有助于突触发育、成熟和稳定的分子过程失调 是很多神经疾病的基础，比如自闭症和精神分裂症形成和长期维持 突触需要许多蛋白质的协同努力-细胞运动因子引导生长锥向突触后生长， 当突触形成时，信号分子会感知，结构蛋白会加强和 稳定发育中的突触然而，分析这些过程的遗传学是困难的，因为许多 这些基因是必需的或多余的。尽管在C. elegans和其他 尽管我们已经建立了模型生物，但关于突触的遗传学仍有许多悬而未决的问题。比如说， 一个生长锥会向哪个方向迁移？一个新形成的突触是如何发出信号 稳定化？这些过程中的缺陷是如何导致疾病病因的？寻找那些 将有助于回答这些问题，使用目前的遗传筛查工具是站不住脚的。我们要求下一个- 新一代的遗传学工具将使我们能够解剖和分析这些过程。 我将开发MosTrap，一种新的基因陷阱诱变方法，可用于正向遗传筛选。 当插入到基因中时，MosTrap转座子使基因失活并表达荧光标记物和荧光标记物。 报告表达模式的标记和可用于选择失活的表型拯救基因 在感兴趣的细胞类型中表达的基因。这种拯救平衡了基因缺失，潜在地使 所有基因的敲除等位基因的产生。使用MosTrap，我将创建一个平衡淘汰赛的集合 神经元基因的等位基因。在创建此集合期间，我将表型敲除菌株的缺陷， 突触功能，以及表征哪些基因是必需的以及每个基因在哪些细胞中表达。 然后我会在这些突变体中筛选出突触稳定性所需的基因敲除。 使用纵向和实时成像，我将在每个应变的突触稳定性缺陷的特点。我们一起努力， 这些数据将确定新的突触稳定性基因，以及告知突触如何在神经元中形成的基础生物学。 变得不稳定，有可能揭示新的机制，可用于治疗自闭症等疾病。 我在这项工作中开发的试剂和方法能够创建一个全基因组的收集， 平衡敲除等位基因。