Worm Breeder's Gazette 15(1): 32 (October 1, 1997)
Department of Biology Lawrence University Appleton, WI 54911
N-ethyl-N-nitrosourea, ENU, is a likely mutagen for genetic studies of C. elegans. We are working to determine optimal buffer and storage conditions in which ENU toxicity remains low with mutagenicity similar to that of EMS. Wild type N2 C. elegans were used in tests of ENU stock and storage conditions. 50 mM ENU stock solutions were prepared in one of four solutions: M9 buffer, 10 mM acetic acid, 10 mM DMSO, and 10 mM Tris pH 6.5. All ENU solutions were diluted to 0.5 or 1.0 mM in M9 buffer and used immediately after dilution. Mutagenesis was for four hours at room temperature. The parent generation of worms did not show any noticeable difference in either lethality or complete sterility, regardless of ENU buffer conditions used. We therefore used Po brood size following mutagenesis as a measure of ENU toxicity. The data show that immediate use of 1.0 mM ENU diluted from a fresh ENU/acetic acid stock provides the highest average brood size at 143 F1 per mutagenized hermaphrodite. This is approximately a two-fold reduction in normal brood size. Results obtained using ENU stock both initially prepared and diluted in M9 are similar, 124 F1/hermaphrodite. Use of ENU stocks prepared in DMSO or Tris appears to be much more toxic as average brood sizes are twenty-fold lower than normal. In an effort to reduce worker exposure to powdered ENU, we undertook experiments to determine whether ENU solutions could be stored without increasing toxicity. Experiments with M9, acetic acid or DMSO as initial solvents indicate that, over time, ENU breaks down to a molecule more toxic to worms. Average brood size drops, sometimes severely, when ENU stock solutions are kept at -20C for as little as two days. ENU stock solutions prepared in Tris were not included in this portion of the study as brood sizes were already remarkably low after immediate use. M9 buffer and DMSO solutions had the largest decreases in brood size. The average brood size resulting from a 1.0 mM ENU solution diluted from an M9 stock dropped from 124, when used immediately, to 39, when mutagenesis was performed after storage for only 2 days at -20C. Use of acetic acid as the initial solvent provided the most suitable environment for storing ENU. After immediate use, the average brood size was 143; after two days of storage at -20C the average brood size had dropped only to 119, a 17% reduction. After eight days of storage, average brood size was further reduced to 34 F1/hermaphrodite. Thus, acetic acid is the optimal solvent for storage of ENU stock solutions for short periods of time only. We previously found that ENU prepared in M9 buffer induces mutations at the same frequency as does EMS. We are currently testing the reversion frequency induced by ENU made from 10 mM acetic acid stocks. In addition, we plan to try reducing the amount of time worms are exposed to ENU.