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

项目总结 促进突触发育、成熟和稳定的分子过程的失调 自闭症和精神分裂症等许多神经系统疾病的基础。的形成和长期维护 突触需要许多蛋白质的协同作用-细胞运动因子引导生长锥体向后 突触细胞，信号分子在突触形成时感觉，结构蛋白加强和 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 这些基因要么是必需的，要么是多余的。尽管在线虫和其他动物中进行了数十年的前向基因筛查 作为模型生物，关于突触的遗传学仍有许多悬而未决的问题。例如, 增长锥体朝向什么信号移动？一个新形成的突触是如何发出它需要的信号的 稳定状态？这些过程中的缺陷如何有助于疾病病因学？寻找基因， 用目前的基因筛查工具来帮助回答这些问题是站不住脚的。我们需要下一步- 新一代基因工具将使我们能够剖析和分析这些过程。 我将开发MosTrap，这是一种新的基因诱变方法，可用于正向遗传筛选。 当插入到基因中时，MosTrap转座子会使基因失活，并表达两种荧光 用于报告表达模式的标记和可用于选择失活的表型救援基因 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 为所有基因创造一个敲除等位基因。使用MosTrap，我将创建一个平衡的敲除集合 神经基因中的等位基因。在创建这个集合的过程中，我将对缺陷的基因敲除菌株进行表型 突触功能，以及哪些基因是必需的，以及每个基因在什么细胞中表达的特征。 然后，我将在这个集合中筛选突变体，寻找突触稳定所需的基因敲除。 使用纵向和实时成像，我将表征每个品系的突触稳定性缺陷。一起， 这些数据将识别新的突触稳定基因，并为突触如何形成的基础生物学提供信息。 变得不稳定，可能会发现可能被用来治疗自闭症等疾病的新机制。 我在这项工作中开发的试剂和方法使创建全基因组集合成为可能 平衡的敲除等位基因。

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.