Washington University in St. Louis School of Medicine
Zebrafish Facilities
WUSTL-SOP-007: ENU Mutagenesis of Spermatogonia in Male Zebrafish
ENU mutagenesis of spermatogonia in zebrafish males
August 17,2004
Lila Solnica-Krezel
Please keep in mind that ENU is a potent alkylating agent, and thus a mutagen. Please exert
extreme caution when dealing with it, keeping in mind both your own safety and of those
around you. Please wear protective clothing and make sure that anything with traces of
ENU on it is decontaminated by incubation in sodium thiosulfate bath.
1. Move fish to be mutagenized in system water to the mutagenesis room 1-2 days
before procedure; adjust salt parameters for system water.
2. Prepare solutions or mutagenesis and recovery;
A. ENU dilution buffer;
1 vol. 95% EtOH
9 vol. 0.1M dibasic sodium phosphate, 0.05M citrate, pH 5.0
B. Mutagenesis and recovery
Egg water (2.4g Instant Ocean in 8L of ddH2O) buffered with 100mM
sodium phosphate to pH 6.5 (pH-meter). Remove water for mutagenesis by filling
x mouse cages with 1L each (plus 1 extra for transfer) and cool down to 20-21oC
on ice. Leave rest in tank and cool down below 19oC on ice for recovery. Add
2.5mL of 4g/L MS-222 (tricaine/MESAB) stock per 1L of recovery water for
anesthetization.
3.
Prepare ENU decontamination tank: dissolve 1,250 g Sodium Thiosulfate in 12.5L of tap
water, buffer with 5M NaOH to pH ~10 (pH-stripes).
4.
Dissolve 1g Isopac ENU by injecting 100mL ENU dilution buffer into the Isopac bottle,
yielding final concentration of ca. 10mg/ml. Keep some dilution buffer left for OD
measurements. (1 – 1.5 hours on the rocker)
You need: 150mL Beaker
60cc Syringe
Needles
Rocker/ tape
All mutagen containing solutions and items should be transferred to sodium thiosulfate bath
and incubated therein for 24 hours.
5. Measure ENU concentration: Dilute 20uL in 1mL of ENU dilution buffer or egg water
(1:50 dilution) and measure OD398 using a disposable plastic pipette.
6. Calculate ENU concentration:
Step1: X mg/ml ENU = OD398 x 69.4
Step2: Molarity can be determined from the proportion, keeping in mind that: 117.3 mg/ml
corresponds to 1000 mM ENU solution
(X mg/ml x 1000mM) / 117.3 mg/ml = YmM
Step3: ENU stock volume needed for 3.5, 3.25 or 3.0 mM of final concentration;
Washington University in St. Louis School of Medicine
Zebrafish Facilities
WUSTL-SOP-007: ENU Mutagenesis of Spermatogonia in Male Zebrafish
(3.0/3.25/3.5 mM x 1000 ml) / Y mM = Z ml
7. Prepare hood for mutagenesis: cover surface with black material (large trash bag), transfer
mutagenesis tanks (20-21oC) to hood, add ENU stock (Z ml) to 1L of mutagenesis buffer
and switch off all lights and noise sources. Transfer fish from a system tank to transfer cage
and very carefully transfer the insert with fish to mutagenesis tank: cover with plastic plate.
Take time immediately and mutagenized for 1 hour. Leave room and prepare recovery
buffer in the mean time (19oC, 10 mg/ml MESAB).
8. After 1 hour of mutagenesis transfer insert with mutagenized fish carefully to tank with
recovery buffer (19oC, 10 mg/ml MESAB). Leave the room. This is the most sensitive step
of the entire procedure!
9. After 2-3 hours transfer fish carefully to fresh recovery buffer (@ RT, no MESAB);
remove any dead fish. Only now empty mutagenesis and recovery buffer into
decontamination tank (sodium thiosulfate) !
10. Next day, change the recovery buffer twice before transferring the males back to the system
in the evening.