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 该对照的分子过程失调。 基于自闭症和精神分裂症等许多神经疾病。 突触需要许多蛋白质蛋白 - 细胞运动因子的共同努力指导生长锥到后 突触细胞，信号分子在形成突触时感应，结构蛋白呈肿胀和 稳定发展的突触。 这些基因是必不可少的或多余的。 模型有机体，仍然是许多开放的问题，例如，突触的遗传学。 生长锥迁移的信号是什么信号 稳定？这些过程中的缺陷如何促进病因？ 将有助于回答这些基因筛查工具，我们需要接下来 - 柳树Ussect的生成遗传工具并分割了这些过程。 我将开发一种新型的基因陷阱诱变方法，可用于正向遗传筛查。 当插入基因时，Mostrap转座子会使基因失活并表达荧光 报告表达模式和表型救援基因的标记，可用于选择失活 基因在这种拯救ballance中表达的基因缺失，有可能使 为所有基因创建淘汰等位基因。 神经元基因的等位基因。 突触功能以及表征哪些基因是必不可少的以及每个基因表达的细胞。 我将在此集合中的筛选中，以获取突触稳定性所需的基因敲除。 使用纵向和实时成像，我将共同表征每个菌株中的突触稳定性缺陷。 这些数据将确定新的突触稳定性基因，并向基本生物学告知突触如何 变得不稳定，可能会发现可能被用来像对待自闭症的新机制。 我在这项工作期间开发的试剂和方法使整个基因组收集的创建 平衡的淘汰赛。