Worm Breeder's Gazette 15(1): 32 (October 1, 1997)

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.

Optimal Buffer and Storage Conditons for ENU

Sara Dorman, Elizabeth De Stasio

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
          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.