Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome.
Double-stranded RNA (dsRNA) mediated gene silencing (RNA interference; RNAi) is a powerful tool for investigating gene function. It is usually performed in Caenorhabditis elegans via the injection or oral delivery of dsRNA, but an alternative approach, the expression of RNA hairpins from introduced DNA (hairpin RNAi; hpRNAi) has several advantages: (1) it can be induced systemically or in a tissue-specific manner; (2) because it is heritable, it allows consistent RNAi silencing across a whole population of genetically identical animals; and (3) it can be applied in refractory tissue such as neurons. hpRNAi has not been widely used to investigate gene function because a number of steps are relatively inefficient and labour-intensive. We describe Wormgate, a new cloning system, which facilitates the efficient high-throughput production of hpRNAi constructs using clones from the C. elegans ORFeome library. The combined use of pWormgate2 and the ORFeome library, with a recently developed particle bombardment transformation system, expedites hpRNAi gene silencing. This will be particularly useful for studying those genes that are refractory to the effects of injected or fed dsRNA, such as neural genes. We report the efficient production of hpRNAi constructs using pWormgate2 and also the knockdown of selected genes, including neurally expressed genes that have previously been refractory to RNAi. Further, when combined with the rrf-3 RNAi hypersensitive strain, the Wormgate approach delivered a highly penetrant knockdown phenotype in nearly 100% of worms for a gene that was completely refractory to other RNAi delivery methods.
Johnson
Nicholas M
NM
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, Australia. Nick.Johnson@anu.edu.au
Behm
Carolyn A
CA
Trowell
Stephen C
SC
D000284:Administration, Oral; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003433:Crosses, Genetic; D005260:Female; D015723:Gene Library; D018398:Homeodomain Proteins / Q000235:genetics; D008297:Male; D009124:Muscle Proteins / Q000235:genetics; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D020543:Proteome / Q000235:genetics; D034622:RNA Interference; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D011978:Receptors, Nicotinic / Q000235:genetics; D015203:Reproducibility of Results; D014170:Transformation, Genetic
D029742:Caenorhabditis elegans Proteins; C487508:Eat-2 protein, C elegans; D018398:Homeodomain Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020543:Proteome; D011978:Receptors, Nicotinic; C073171:unc-4 protein, C elegans; D012324:RNA-Dependent RNA Polymerase; C472756:RNA-directed RNA polymerase RRF-3, C elegans