Series 3: Reverse Genetics using RNAi in C. elegans
In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects is that we study a defective gene to find out how its product is functionally defective and then we must infer the function of the normal form of the gene. Ultimately, we care most about what that mutation can tell us about normal gene function. In reverse genetics projects we start with a normal gene rather than a mutant phenotype and we somehow prevent the normal gene from producing it's product to find out the function of that gene and gene product. In the past, that gene disruption was done by creating a "knockout": an organism that had the gene deleted but was still viable for study. Most commonly this was done in mice but these knockout mice were VERY expensive and difficult to produce and maintain.
In recent years, the usefulness of the C. elegans model system has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi). 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 Center 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.
Links to Labs& Project Info
Lab 11: RT PCR reactions
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Schedule of Reverse Genetics Project
RNAi General Information
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification