N-Nitroso-N-ethylurea ISOPAC (Sigma N3385) containing 1 g powder, stored at -20°C

95% EtOH (not made from absolute)

Phosphate-citrate buffer (pH 5.0-6.0)

0.1 M NaH₂PO₄

0.05 M Na₃C₆H₅O₇

5% Na₂S₂O₃ in 0.1 M NaOH (to inactivate ENU)

20-gauge needles, 10 mL and 60 mL syringes for solution prep

a beaker (container) with warm water for solution prep

1 mL insulin syringe for injections

double gloves, face shield for injections

spectrophotometer (Beckman DU530)

spectrophotometer cuvettes (Brand Tech Scientific 759200, Ultramicro size, 8.5 mm window height)

ENU preparation and injections are to be done in a high flow fume hood.

ENU preparation

1. Inject 10ml of 95% EtOH using a 20-gauge needle and 10 mL syringe into the ISOPAC.  Discard the needle and syringe.

2. Swirl the ISOPAC in the warm water bath until ENU powder is fully dissolved.

3. Add 90 mL of room temperature Phosphate-citrate buffer using 60 mL syringes and 20-gauge needle.

Note: Since ENU is easily degraded, the precise concentration should be determined based on absorbance using a spectrophotometer at λ=398 nm.  Concentration is calculated based on the formula 0.72 O.D. = 1 mg/ml of ENU.

Dosage for mutagenesis

-3 weekly doses of 100 mg/ml.

-Generally the mutagenized males will recover from sterility at week 12.  A test-breed can be performed 8 weeks after the last ENU injection to monitor the recovery of fertility.

ENU injections and clean-up

1. Prepare the work surface in the hood with diapers.

2. Wear double gloves and face shield and perform the injection in the fume hood.

3. Weigh all of the mice to be injected.

4. Inject each mouse i.p. with 100 mg ENU per kg body weight.  Place injected mice in disposable cages to be left in hood overnight.

Clean-up

5. Inactivate the remaining ENU by injecting 5% Na₂S₂O₃  to the ISOPAC.

6. Spray work area, especially any regions of potential ENU spills with 5% Na₂S₂O_3.

7. The next morning, transfer mice back to normal facility. Discard the disposable cages and diapers as biohazard waste.