The “Feeding” Protocol
Bacteria can be engineered to overexpress dsRNA, and since the laboratory diet for C. elegans is bacteria, this forms the basis of the “feeding protocol”, an ingestion-based dsRNA delivery method. The “feeding protocol” can be used to deliver dsRNA to other organisms that normally feed on bacteria (hydra, for example). Other groups have mixed dsRNA-expressing bacteria with the typical food for the organism (for example, dsRNA-expressing bacteria can be mixed with liver and fed to planaria). Variations in this protocol may one day be used to treat human disorders. The feeding protocol is an inexpensive and relatively easy way to demonstrate modern reverse genetics techniques and to help make the phenotype:genotype connection in high school and undergraduate laboratory courses. Here we describe how we perform the feeding protocol in our lab. The pdf file below has ordering information for strains and plasmids.
Equipment, Supplies, and Reagents; Solutions; Procedures
- Prepare dsRNA expressing bacteria Isolate dsRNA-expressing plasmid from bacteria Re-transform competent HT115(DE3) cells with plasmid and plate. After one week, re-transform for best results.
- Inoculate overnight culture
- Dilute culture and induce. (We induce in culture, spiking with Tet+Amp+IPTG three times over the course of a few hours Seed NGM plates supplemented with Tet+Amp+IPTG. Let dry overnight.
- Place C. elegans onto seeded plates. We usually add L1 larvae and incubate at 22-25 degrees C for 2-5 days.
- Monitor phenotype daily
An example of an RNAi phenocopy. The unc-22 gene, when mutated, causes the animals to twitch. Twitching can be oberved in mutants (left) as well as dsRNA-treated animals (right).
- unc-22 mutants
(genetic defect in unc-22 gene
- RNAi-defective mutants
(unable to respond to dsRNA)
- unc-22 RNAi phenocopy
(wild-type animals ingesting unc-22 dsRNA)
• Timmons, L. (2006) Construction of plasmids for RNA Interference and in vitro transcription of double-stranded RNA. C. elegans: Methods and Applications. (Humana Press) Kevin Strange, ed., pp. 109-118.
• Timmons, L. (2006) Delivery methods for RNA interference in C. elegans. C. elegans: Methods and Applications. (Humana Press) Kevin Strange, ed., pp. 119-125.
• Hull, D. and Timmons, L. (2004) “Methods for delivery of double-stranded RNA into C. elegans” in Methods Mol Biol: RNA interference, editing, and modification. (Humana Press) Jonatha Gott, ed. 265:23-58.
• Timmons, L. and Fire, A. (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103-12.
• Timmons, L. and Fire, A. (1998) Specific genetic interference from ingested double-stranded RNA. Nature 395(6705):854.
• Lohmann, J.U., Endl, I., and Boxch, T.C.G. (1999) Silencing of Developmental Genes in Hydra. Developmental Biology 214:211-214.
• Newmark, P. A., Reddien, P. W., Cebria, F., and Alvarado, A. S. (2003) Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. PNAS 100:11861-11865.
• Xiang, S., Fruehauf, J. and Li, C. J. (2006) Short hairpin RNA-expressing acgteeria elicit RNA interference in mammals. Nature Biotechnology 24(6):697-702.