Bipartite gene expression system for C. elegans genetic and neural circuit analysis

用于线虫遗传和神经回路分析的二分基因表达系统

• 批准号: 9437389
• 负责人: PAUL Warren STERNBERG
• 金额: $ 24.75万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-18 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9437389
• 关键词: Address; Adoption; Animal Model; Animals; Back; Behavior; Binding; Biological Assay; Caenorhabditis elegans; Cells; Communities; Complex; DNA Binding Domain; Data; Development; Documentation; Dorsal; Drosophila genus; Exercise; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Research; Goals; Growth; Head; Histamine; Individual; Instinct; Mediating; Methods; Modeling; Modification; Molecular; Molecular Analysis; Monitor; Motor; Nature; Nervous system structure; Neurobiology; Neurons; Organism; Partner in relationship; Pheromone; Play; Process; Protein Splicing; Proteins; Reagent; Research Personnel; Role; Scheme; Sensory; Series; Side; System; Tail; Temperature; Testing; Tetanus Toxin; Tissues; Transcription Coactivator; Transcriptional Activation Domain; Transgenes; Vertebral column; Vulva; Yeasts; calcium indicator; cell type; expression vector; gene function; genetic analysis; improved; intein; interest; male; mating behavior; mutant; neural circuit; neuronal circuitry; optogenetics; promoter; reconstitution; relating to nervous system; sensor; sperm cell; systems research; tool

Project Summary Understanding the molecular and cellular basis for behavior depends on a rigorous assessment of the contributions of different neuron types. Progress in elucidating neural circuits for behaviors is often hampered by a lack of genetic tools for efficiently generating animals with cell-type specific expression of genes that can be used to perturb or monitor neuronal activity, such as optogenetic tools, tetanus toxin, and GCaMP. Also, there has been steady progress in optogenetics and genetically-encoded sensors such as GCaMP calcium indicators, but it is impractical to rebuild hundreds of strains inserting each improved version into a repertoire of expression vectors. One elegant method that addresses both issues simultaneously is to use a bipartite expression system, which separates the cell-type control from the effector, and thus a set of cell-type specific drivers can be reused with different versions of effectors (e.g., GCaMP6 versus GCaMP3). Conversely, a set of strains might be made with a promoter that directs expression in two or more cell types; if a more specific regulatory sequence is identified, then all the constructs have to be rebuilt. With a bipartite system, construction of a single Driver with the new regulatory sequence can easily combined with all the available Effectors to efficiently generate the strains needed. allows use of all the Effectors. For example, Drosophila researchers have made great use of the Gal4-UAS system in which a transcriptional activator protein (Gal4) is expressed in the cell type(s) of interest and binds to its target sequence – the UAS – to direct expression of an effector gene of interest. This scheme allows many combinations of specifically expressed genes to be built from a much smaller number of transgenes. However, Caenorhabditis elegans has not had such a system until our recent development of the cGAL system, an optimized Gal4-UAS system. One key feature of our implementation is the use of the DNA-binding domain of the Gal4 protein from a yeast species whose optimal growth temperature matches that of C. elegans, thereby allowing more efficient target gene activation. We also showed that the cGAL system can be applied to functional studies in C. elegans. We propose to construct an initial neuronal cGAL toolkit, and apply it to one circuit as proof of principle. The chosen circuit is male mating behavior, arguably the most complex of C. elegans behaviors as it involves almost the entire nervous system and a complex series of steps each involving sensory-motor integration. While the roles of many male specific neurons have been identified, the roles of non-sex-specific neurons have not; our approach will make the cGAL reagents that render all of the non-sex-specific neurons tractable to analysis in a systematic way. At the end of two years, we will have fully introduced a useful bipartite expression system to the C. elegans community and refined our understanding of innate behavior.

项目摘要 了解行为的分子和细胞基础取决于对 不同神经元类型的贡献。阐明行为的神经元电路的进展常常受到阻碍 缺乏有效地产生具有细胞类型的基因表达的动物的遗传工具 用于扰动或监测神经元活性，例如光遗传学工具，Tetanius毒素和GCAMP。另外，那里 在光遗传学和遗传编码的传感器（例如GCAMP钙指标）中一直是稳定的进展， 但是，重建数百种改进版本的应变是不切实际的，这是不切实际的 向量。解决这两个问题的一种优雅方法是使用二分表达系统， 将细胞类型的控制与效应子分开，因此可以重复使用一组细胞类型的驱动程序 具有不同版本的效果（例如，GCAMP6与GCAMP3）。相反，可能会产生一组菌株 带有指导两种或多种细胞类型表达的启动子；如果更具体的调节序列是 确定的，然后必须重建所有结构。使用双方系统，建造一个驾驶员 新的调节序列可以轻松地与所有可用效应子结合在一起，以有效地产生 需要菌株。允许使用所有效应子。例如，果蝇研究人员充分利用了 GAL4-UAS系统，其中转录激活蛋白（GAL4）在感兴趣的细胞类型中表达 并结合其目标序列 - UAS - 直接表达感兴趣的效应基因。这个方案 允许从少量翻译中构建许多特殊表达基因的组合。 但是，直到我们最近开发CGAL系统，秀丽隐杆线虫才有这样的系统， 优化的GAL4-UAS系统。我们实施的一个关键特征是使用DNA结合域 来自酵母菌的GAL4蛋白，其最佳生长温度与秀丽隐杆线虫相匹配，从而 允许更有效的靶基因激活。我们还表明，CGAL系统可以应用于功能 秀丽隐杆线虫的研究。我们建议构建一个初始神经元CGAL工具包，并将其应用于一个电路作为证明 原则。选定的电路是男性交配行为，可以说是秀丽隐杆线虫行为最复杂的行为 它几乎涉及整个神经系统和一系列复杂的步骤，每个步骤涉及感觉运动 一体化。虽然已经确定了许多男性特异性神经元的作用，但非性质特异性的作用 神经元还没有；我们的方法将使CGAL试剂呈现所有非性质特异性神经元 可以以系统的方式进行分析。在两年结束时，我们将充分引入一个有用的两部分 秀丽隐杆线虫社区的表达系统，并完善了我们对先天行为的理解。