By Bruce Nash, PhD
Dolan DNA Learning Center, Cold Spring Harbor Laboratory /
Many genes—no function
As DNA sequencing has become faster and cheaper, scientists have been able to identify genes at an ever-accelerating pace. The function of many of these genes is unknown. Recently, the discovery of a mechanism called RNA interference (RNAi) has provided just the tool that scientists need to quickly determine the function of genes. In my previous article, I introduced the model organism C. elegans. In this article, I am introducing an experiment that lets students turn off genes in C. elegans using RNAi.
The discovery of RNAi
In 1990, while working for the biotech firm DNA Plant Technology Inc., Rich Jorgensen and Carolyn Napoli tried to make petunias more purple than normal. Engineering petunias was meant to show potential investors that the firm had the technology to manipulate commercially important flowers. To do so, the scientists introduced extra copies of the gene encoding an enzyme called chalcone synthase (CHS) into petunia cells. This enzyme is part of the biosynthetic pathway that makes anthocyanins, the purple pigments in plants. They predicted that increasing the number of CHS genes would increase CHS protein levels, leading to the production of very purple flowers.
Instead, introducing the CHS gene had the opposite effect: rather than being darker, many of the modified flowers were white or had white variegations. How could this happen? When Jorgensen and Napoli looked in more detail, they found that the levels of CHS messenger RNA (mRNA) in white flowers were 50 times lower than in unaltered plants. Somehow the introduced gene (the transgene) lowered expression of both the transgene and the endogenous petunia gene. Jorgensen’s petunias were just one example of this phenomenon; unexpected silencing (reduced gene expression) also happened when scientists did similar experiments in animals, other plants, and fungi.
Inducing RNAi by
Feeding Kit
Culturing and Observing C. elegans Kit
How silencing occurred was a mystery for almost 10 years. One popular model suggested that silencing was caused by anti-sense RNA—single-stranded RNA that is the reverse complement of mRNA. The idea was that anti-sense RNA would base pair to the message and inhibit protein production. In 1998, Andrew Fire and Craig Mello discovered that injecting C. elegans with double-stranded RNA (dsRNA) with a sequence corresponding to a gene would silence that gene.
In fact, dsRNA was more than 100 times more effective at inhibiting gene function than anti-sense RNA. When scientists checked other examples of silencing, including Rich Jorgensen’s petunias, they found that dsRNA complementary to the silenced gene was produced in each case, showing that this response occurs in multiple organisms. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for discovering RNA interference—the term they coined to describe gene silencing by dsRNA.
RNAi is routinely used to silence genes and study their function. Its power comes from the ability to quickly target genes in a sequence-specific way. Also, RNAi promises to give rise to a new family of RNA-based drugs, with treatments being developed for cancers, neurodegenerative diseases like Alzheimer’s, and viral infections like HIV and hepatitis. Likewise, RNAi is being explored as a way to engineer new crops. Examples of genetically engineered crops using RNAi technology include tobacco with reduced nicotine levels; soybeans with better oil quality; and papaya, plum, and squash with viral resistance.
Inducing RNAi by feeding
Amazingly, feeding the roundworm C. elegans bacteria expressing dsRNA identical to a worm gene of choice can silence that gene. The experiment is very simple to set up. First, 3 strains of bacteria are grown: control bacteria and 2 strains designed to “silence” 2 different genes. These strains have been engineered to express dsRNA complementary to each targeted gene. Next, wild-type worms are fed each bacterial strain. The control bacteria have no effect. However, when worms eat bacteria expressing dsRNA, it is taken up by the worms and recognized by the RNAi machinery, turning off the targeted gene. Finally, once progeny from the worms grow up, students can observe the phenotypes with a dissecting microscope, compare the control worms to the RNAi-treated worms, and hypothesize about the function of the silenced genes.
To learn more about the genes in question, students explore them in the C. elegans WormBase database. Once they have identified the sequence for the genes, they are guided in using the Basic Local Alignment Search Tool (BLAST) bioinformatics Web site to identify human homologs of these genes.
This experiment provides a great educational opportunity: you can have your students turn off genes and see the results just days later. It is a vivid demonstration of the relationship between genes, their function, and phenotypes using Nobel Prize-winning technology.
Author’s note: The RNAi experiment discussed can be done with your students using Carolina’s Inducing RNAi by Feeding Kit. Carolina also offers the Culturing and Observing C. elegans Kit containing naturally occurring C. elegans mutants with the same phenotype created using RNAi in the Inducing RNAi by Feeding Kit.
“RNA Interference: Turning Genes Off at Will, Part II” will appear in a future issue of Carolina Tips®. Don’t miss it!—Ed.