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 不同神经元类型的贡献。在阐明行为的神经回路方面的进展经常受到 缺乏遗传工具来有效地产生具有细胞类型特异性表达的基因的动物 用于干扰或监测神经元活动，如光遗传工具、破伤风毒素和GCaMP。还有，还有 在光遗传学和GCaMP钙指示剂等遗传编码传感器方面取得了稳步进展， 但是，重建数百个菌株并将每个改进的版本插入到表达谱中是不切实际的 向量。同时解决这两个问题的一种优雅的方法是使用两部分表达系统， 其将细胞类型的控制与效应器分开，从而可以重复使用一组细胞类型的特定驱动器 使用不同版本的效应器(例如，GCaMP6与GCaMP3)。相反，可能会产生一组菌株。 具有在两种或两种以上细胞类型中指导表达的启动子；如果更特定的调节序列是 识别出来，那么所有的构造都必须重建。在两部分系统中，构建具有 新的调节序列可以很容易地与所有可用的效应器相结合，以有效地产生 需要的菌株。允许使用所有效应器。例如，果蝇研究人员很好地利用了 Gal4-UAS系统，其中转录激活蛋白(Gal4)在感兴趣的细胞类型(S)中表达 并与其靶序列UAS结合，直接表达感兴趣的效应器基因。这项计划 允许用数量少得多的转基因构建多种特定表达的基因组合。 然而，直到我们最近开发出cGAL系统，秀丽线虫才有这样的系统， 一种优化的Gal4-UAS系统。我们实现的一个关键特性是使用DNA结合域 来自一种最适生长温度与线虫相匹配的酵母物种的Gal4蛋白，从而 从而允许更有效的靶基因激活。我们还证明了cGAL系统可以应用于功能 对线虫的研究。我们建议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 原则性的。所选择的电路是雄性交配行为，可以说是线虫最复杂的行为，如 它几乎涉及整个神经系统和一系列复杂的步骤，每个步骤都涉及到感觉-运动 整合。虽然许多男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 神经元没有；我们的方法将使cGAL试剂使所有非性别特异性神经元 易于系统地分析。在两年结束时，我们将完全引入一个有用的两方 表达系统对线虫群落和提炼我们的先天行为的理解。

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.