ABLE awarded a Roberta Williams Laboratory Teaching Initiative Grant last summer for improvements to a laboratory that focuses on student designed experiments that test the longevity of nematodes. The laboratory is designed for first or second year undergraduate students, although it could be adapted for more advanced levels.
Aging is a common feature of animals that have a separate soma and germ line. Both environmental and genetic factors are responsible for the changes categorized as aging such as accumulation of damaged cells, decreased physiological capacity, and increased susceptibility to disease. Many animal models exist to test hypotheses of aging, from brewers yeast to mice. However, the free-living nematode Caenorhabditis elegans is one of the best organisms to use for simple, short-term longevity experiments for many reasons. C. elegans has a normal lifespan between 2-3 weeks, is non-pathogenic, is easy to maintain, can be manipulated under a stereo microscope, and exhibits behaviors and physical manifestations of aging that parallel those of more complex animals. An important component of the aging lab is that it allows students to develop and test their own hypotheses of environmental and genetic factors affecting C. elegans aging. The lab itself involves placing worms of known age on selective agar plates and recording their appearance and survival for a few weeks.
Important design features to add to the laboratory
To simplify the set up and maintenance required to provide this lab, I am creating new strains of C. elegans that have temperature controlled sterility and increased sensitivity to a post-translational gene regulation effect called RNA interference (RNAi). The temperature controlled sterility, conferred by a fem-1 allele, is essential for observing individual worms in a population without the confounding effects of additional generations. If the worms were allowed to reproduce, overpopulation and starvation of a culture would occur in as little as a week, a timeframe much shorter than the lifespan of individual worms. Elimination of progeny complicated previous versions of this lab. In addition, adding RNAi susceptibility by including the rrf-3 allele enables gene-specific loss-of-function phenotypes to be obtained just by feeding the worms E. coli containing plasmids encoding C. elegans genes. This allows students to test phenotypes not available genetically by placing special E. coli on the agar plates as food.
In March 2008, I will test the new additions to the C. elegans Aging lab. These include the newly created genetic strains, daf-16 I; rrf-3 II, nhr-49 I; rrf-3 II, sod-3 X; rrf-3 II, and fat-7 V; rrf-3 II. A second set of genetic crosses to add the temperature sensitive fertility gene, fem-1, into these strains is underway. As an alternative to fem-1, I am including a chemical called FUDR (floxuridine) in the C. elegans media. FUDR inhibits DNA synthesis, which has little effect on adult worms and longevity, but inhibits embryo development and hatching. While the amount of FUDR in the media is very low (25 um, or ~.025 mg per liter), the forthcoming fem-1 temperature sensitive strains should eliminate the necessity of its inclusion. Because the genetic crosses to create triple mutant strains such as daf 16 I; rrf-3 II; fem-1 IV do not result in easily detectable phenotypes; I will be using PCR to identify animals homozygous at each loci. Once completed, this lab should provide students with robust results while testing the interactions between the environment and aging genes.
I look forward to presenting the completed lab at the ABLE 2009 meeting and would like to thank the members of ABLE for their support.
Pliny A. Smith
Lake Forest College