Silencing Genomes

With complete genome sequences accumulating at an accelerating rate, ahead lies the massive task of determining the physiological function of thousands of newly identified genes for which little is known beyond their sequences. The 2006 Nobel Prize in Physiology or Medicine recognized the discovery RNA interference (RNAi), a basic mechanism of gene regulation that also provides an important new tool for functional genome analysis. In RNAi, short, double-stranded RNA molecules can down-regulate gene expression of a corresponding target gene. By deliberately introducing defined sequences of dsRNA into living organisms, biologists can observe the physiological consequences of "silencing" virtually any gene in C. elegans, as well as other plants and animals.

Despite its power, RNAi is amazingly simple to perform in the roundworm C. elegans, an important model system for eukaryotic gene function. Any gene of choice can be "silenced" merely by feeding worms bacteria that express the correct double-stranded RNA. In its simplest form, RNAi requires little more than the ability to grow bacteria and observe C. elegans traits with a dissecting microscope. The vast majority of high schools and colleges meet these requirements, making RNAi in C. elegans potentially more accessible than other molecular techniques for which specialized equipment is required - such as PCR and gel electrophoresis. For these reasons, we have devoted considerable effort to developing the RNAi/C. elegans system as the vehicle to deliver functional genome analysis into high school and college classes.

The Silencing Genomes site is part of a National Science Foundation project to develop an integrated experiment- and bioinformatics-based curriculum on RNAi in C. elegans. The curriculum begins with observation of mutant phenotypes and basic worm "husbandry," then progresses to simple methods to induce RNAi and to use RNAi to rescue (compensate) a mutant phenotype. A more advanced experiment uses "single-worm PCR" to examine the mechanism of RNAi - comparing the DNA of worms with identical phenotypes induced either by RNAi or a gene mutation. The curriculum culminates with a open-ended methods that support student projects. Students can perform RNAi "from scratch" using bioinformatics to develop PCR primers for a target gene, then cloning the amplified product into an RNAi feeding vector, and finally observing the phenotype of treated worms. Students also have free access to the DNALC's collection of RNAi feeding strains, which can be used to conduct a mini-screen to identify genes involved in a particular biological pathway.

An online lab notebook, Silencing Genomes combines lab methods with user-friendly features adapted from the DNALC's popular text DNA Science - including flow charts, reagent recipes, and extensive instructor information. Supporting resources include photos and video of C. elegans mutants, as well as a simple check-out system to obtain any of 80 C. elegans mutants and E. coli feeding strains. The Internet site also provides a launch pad for bioinformatics exercises that accompany each experiment. Students use online databases - including WormBase and Pubmed - to explore the molecular genetics and physiological functions of the genes targeted by RNAi. NCBI's BLAST and the DNALC Sequence Server are used to explore the evolutionary relatedness of genes in worms and humans.

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