This website was created as an assignment for Genetics 564 at the University of Wisconsin-Madison
RNAi RNA interference or RNAi is a research technique used to knockdown genes without changing the genetic makeup of the organism. Popularized in the model organism C. elegans, RNAi has become a useful tool for determining the function of a gene in reverse genetics. In C. elegans, the technique involves feeding the worm double stranded RNA (dsRNA) through a bacterial plasmid vector. This dsRNA is then processed by a series of enzymes which eventually uses the dsRNA as a template to find the mRNA transcribed by the gene of interest. Once found, these enzymes destroy the mRNA transcript before its able to to be translated into a protein. This results in a gene silencing phenotype [1]. The figure on the right shows the mechanism for gene silencing via RNAi. |
RNAi and the RYR2 Gene
The C. elegans homologue for the RYR2 gene can be given RNAi to knock down Unc-68, the specific gene orthologous to RYR2. Using WormBase, we can see that many Unc-68 RNAi knockdowns have been performed. Two phenotypes seen as a result from an RNAi knockdown of Unc-68 are aldicarb resistance and locomotion varaibility[2]. Aldicarb is an insecticide that is specifically used to kill nematodes. It is a cholinesterase inhibitor which prevents the breakdown of acetylcholine and leads to neural poisoning[3]. The locamotion variant from RNAi knockdown shows that Unc-68 is involved in muscle contraction[2]. I also searched for RNAi phenotypes for mice and fruit fly using Mouse Genomic Informatics and Flybase respectably, but no RNAi phenotypes were found.
Analysis of RNAi
It is no surprise that a knockdown of Unc-68 via RNAi causes a locomotion variant since Unc-68 acts a calcium ion channel in muscle tissue of C. elegans. It is surprising though that muscle contraction is even possible with a complete knock down. This could suggest that either Unc-68 isn't the only protein regulating calcium ion concentration in the muscle tissue or that the RNAi isn't capable of completely knocking down the gene since muscle contraction is still occurring. For mice, I was not surprised that no RNAi phenotypes were found for the RYR2 protein. First, RNAi is very difficult to perform in mammals and often cannot performed en vivo. Also, I would suspect that a complete knockdown of RYR2 would cause lethality since it is expressed in early development of the heart and the brain. A complete knockdown would likely cause developmental issues that would not allow the mouse to survive.
References:
[1]Kamath, Ravi S. et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 421, 231-237.
doi:10.1038/nature01278 http://www.nature.com/nature/journal/v421/n6920/full/nature01278.html
[2] Wormbase for Unc-68. Retrieved April 21, 2014, from http://www.wormbase.org/species/c_elegans/gene/WBGene00006801?query=unc-
68#0acb-9d6-3
[3] Becker, Dan. (2010). Aldicarb. Toxipedia. Retrieved April 23, 2014 from http://www.toxipedia.org/display/toxipedia/Aldicarb
RNAi Image- http://www.intechopen.com/books/practical-applications-in-biomedical-engineering/structure-functions-relations-in-smallinterfering-rnas
[1]Kamath, Ravi S. et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 421, 231-237.
doi:10.1038/nature01278 http://www.nature.com/nature/journal/v421/n6920/full/nature01278.html
[2] Wormbase for Unc-68. Retrieved April 21, 2014, from http://www.wormbase.org/species/c_elegans/gene/WBGene00006801?query=unc-
68#0acb-9d6-3
[3] Becker, Dan. (2010). Aldicarb. Toxipedia. Retrieved April 23, 2014 from http://www.toxipedia.org/display/toxipedia/Aldicarb
RNAi Image- http://www.intechopen.com/books/practical-applications-in-biomedical-engineering/structure-functions-relations-in-smallinterfering-rnas
Site created by: Mercede Davis
Email contact: [email protected]
Date last updated: 5/14/14
Genetics 564, University of Wisconsin-Madison
Email contact: [email protected]
Date last updated: 5/14/14
Genetics 564, University of Wisconsin-Madison