Worm Breeder's Gazette 15(4): 18 (October 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Dept. of Biology Lawrence University, Appleton, WI
Our lab previously reported the mutagenic spectrum of ENU using reversion of unc-93(e1500) (Genetics 147:597). We found that A/T->G/C or A/T->C/G changes comprised half of the sequenced ENU-induced point mutations. It is clear that this broader mutagenic spectrum could make ENU a superior mutagen for genetic studies requiring, for example, non-null alleles. The major drawback to ENU mutagenesis, however, is its relatively high, concentration-dependent toxicity to C. elegans. In recent experiments, we sought to reduce both ENU toxicity to worms and worker exposure to powdered ENU. We assessed worm toxicity by measuring brood sizes of mutagenized L4/young adult hermaphrodites. ENU is known to degrade in aqueous solutions, thus we tested a variety of solvents for the production of 50 mM ENU stock solutions, including: 10 mM acetic acid, 1X M9 buffer, 10 mM DMSO, 10 mM Tris buffer, and 100% ethanol. In all cases a 50 mM ENU stock was diluted to 0.3 or 0.6 mM in M9 buffer for a 4 hour mutagenesis. Drastic decreases in brood sizes were seen at one or both ENU concentrations when ENU was used from stocks made in M9 buffer, DMSO, or Tris buffer. Brood sizes remained stable at an acceptable 70% of normal levels when ENU was diluted from stocks made in acetic acid or 100% ethanol. Ethanol stock solutions were found to be much more stable, however. Aliquots of ENU stocks were stored at -20C and used for mutagenesis experiments. Brood sizes of animals exposed to ENU from a 15-day old acetic acid stock solution decreased to <10% of normal levels; ENU/ethanol stock solutions could be similarly stored with little effect on brood size of animals. Lastly, we tested the mutagenicity of ENU diluted from a 50 mM ENU/ethanol stock solution. Using reversion of unc-93(e1500), we found the reversion frequency to be 2.6 X 10-4, not significantly different from that reported previously using ENU from an ENU/M9 stock solution diluted immediately after preparation. We suggest the C. elegans community adopt a standard procedure for ENU mutagenesis in which the following methods are used: 1. ENU stock solutions are prepared in fresh 100% ethanol. Care is taken to note the water content of each lot of ENU and adjust amounts used to provide a consistent ENU concentration between lots. Such stock solutions may be kept at -20C and used for up to 2 weeks with little increase in toxicity and no noticeable change in mutagenicity. 2. Mutagenic solutions are best made in the range of 0.5 to 3.0 mM ENU in M9 buffer (Lewis & Fleming, 1995 version). The concentration used may depend on the health of the strain to be mutagenized. The volume of mutagenic solution used is 4 mls for a healthy worm population from a single 60 mm plate. Mutagenesis is done at room temperature for 4 hours with gentle agitation (50 rpm on a rotary shaker). Following mutagenesis, worms are washed twice with M9 buffer and placed on growth media. 3. All ENU solutions should be rendered inactive before being discarded. This may be done by high pH and treatment with sodium thiosulfate. Solutions can be brought to a final concentration of 10% sodium thiosulfate, 1% sodium hydroxide and kept at room temperature for one hour (6 half-lives) (The Zebrafish book at http://zfish.uoregon.edu/zf_info/zfbook/zfbk.html).