BISC 219: Mod 2 RNAi General Information

In recent years, the usefulness of the C. elegans model system has been dramatically enhanced because it is particularly suited to gene silencing by RNA interference (RNAi). In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. However, RNAi disrupts gene expression in an entirely different way – by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell. By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in C. elegans, as well as many other plants and animals. Amazingly, this mechanism can be activated in C. elegans by simply feeding worms bacteria expressing dsRNA that corresponds to part of the gene to be silenced. An altered phenotype in the progeny of RNAi-treated worms indicates what happens when the normal function of this gene is lost. The other two methods of RNAi in C. elegans are the soaking method, in which animals are soaked in dsRNA, and the injection method, in which dsRNA is microinjected into worms. How does this happen in the worms? The enzyme dicer recognizes dsRNA and degrades (or cuts) it into siRNA (small interfering RNA), which is then taken into the RISC complex that degrades mRNA sequences that are identical (or close to identical) to the siRNA. As a historical sidelight, although previously observed in a number of other organisms, RNAi was truly developed using C. elegans. This resulted in a 2006 Nobel Prize to Craig Mello at UMass Medical in Worcester, MA and Andy Fire at Carnegie Mellon, for their research in this area. This is one of 3 Nobel Prizes won using C. elegans as a model organism! I could recreate the history of RNAi here to explain it but many more people have done it better than I ever could. Here is a link to a great overview of RNAi and its history from Ambion Biosciences RNAi Pages. Please examine The Overview of RNA interference and The Mechanism of RNA interference. This is a great beginning to understanding RNAi not only in non-mammalian cells but also the differences between non-mammalian and mammalian gene silencing. You might also want to check out NOVA Science Now RNAi Explained. Here is a link to animations from Nature: Animations.

RNAi Schedule of Experiments Media Recipes Lab 4: Picking your gene to RNAi Lab 5: Plasmid DNA isolation and transformation Lab 6: Induction of RNAi plasmid and C. elegans feeding Lab 7: Single Worm PCR and Agarose Gel Electrophoresis Lab 8: PCR Reaction Cleanup and Sequencing