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

项目摘要 理解行为的分子和细胞基础取决于对行为的严格评估。 不同神经元类型的贡献。阐明行为的神经回路的进展经常受到以下因素的阻碍： 缺乏有效产生具有细胞类型特异性表达基因的动物的遗传工具， 用于干扰或监测神经元活动，如光遗传学工具，破伤风毒素和GCaMP。而且 在光遗传学和遗传编码传感器如GCaMP钙指标方面取得了稳步进展， 但是将每一个改良的版本插入到一个表达库中来重建数百个菌株是不切实际的 向量。一个同时解决这两个问题的优雅方法是使用二分表达式系统， 其将细胞类型控制与效应分离，并且因此可以重复使用一组细胞类型特异性驱动器 对于不同形式的效应器（例如，GCaMP 6对GCaMP 3）。相反地，一组菌株可能会被制造出来， 具有指导在两种或更多种细胞类型中表达的启动子;如果 一旦确定，所有的结构都必须重建。使用二分系统， 新的调节序列可以容易地与所有可用的效应子组合， 需要的应变。允许使用所有效应器。例如，果蝇研究人员已经充分利用了 Gal 4-UAS系统，其中转录激活蛋白（Gal 4）在感兴趣的细胞类型中表达 并结合其靶序列-UAS -以指导感兴趣的效应基因的表达。该方案 允许从更少数量的转基因构建特异表达基因的许多组合。 然而，秀丽隐杆线虫还没有这样的系统，直到我们最近开发了cGAL系统， 优化的Gal 4-UAS系统。我们的实现的一个关键特征是使用的DNA结合域的 Gal 4蛋白来自最适生长温度与C.因此， 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性 C.优雅我们提出构建一个初始的神经元cGAL工具包，并将其应用于一个电路作为证明 的原则。选择的回路是雄性交配行为，可以说是C中最复杂的。优雅的行为， 它几乎涉及整个神经系统和一系列复杂的步骤，每个步骤都涉及感觉运动， 一体化虽然许多雄性特异性神经元的作用已经被确定，但非性别特异性神经元的作用仍然存在。 神经元没有;我们的方法将使cGAL试剂，使所有的非性别特异性神经元 易于系统分析。在两年后，我们将全面引入一个有用的双边 表达系统与C. elegans社区和完善我们的理解先天行为。

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.