Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function.
Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we 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 GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We 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.
Department of Genetics, Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, Connecticut, United States of America.
Christopher
C
Firnhaber
Marc
M
Hammarlund
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000254:growth & development; D002875:Chromosomes / Q000235:genetics; D004298:Dopamine / Q000235:genetics / Q000378:metabolism; D059330:GABAergic Neurons / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D020043:Genes, Essential; D009420:Nervous System / Q000254:growth & development / Q000378:metabolism; D034622:RNA Interference; D005680:gamma-Aminobutyric Acid / Q000235:genetics*
D005680:gamma-Aminobutyric Acid; D004298:Dopamine