Neuron-specific RNAi reveals neuronal functions of essential genes.

摘要

Living organisms often reuse the same genes multiple times for different purposes. If one function of a gene is essential, death or arrest of the mutant masks other functions. Understanding the functions of essential genes is particularly critical in the nervous system, which must maintain its form and function long after development is complete. However, current strategies for generating conditional knockouts rely on making a new transgenic animal for each gene and thus are not useful for forward genetic screens or for other experiments involving a large number of genes.;I have developed a technique in C. elegans for generating gene knockdown in selected neuron sub-types in response to feeding RNAi. I combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. I produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABAergic, cholinergic, dopaminergic, or glutamatergic neurons. In these strains, I observe neuron cell-type specific behavioral changes when I knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes.;Using the GABAergic neuron-specific RNAi strain, I screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and Ill for their effect on GABA neuron function. I identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.