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.

Further Optimization of ENU Mutagenesis

Beth De Stasio, Sara Dorman

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

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