- The Jackson Laboratory

Mouse Mutagenesis with ENU

Material and Methods:

N-ethyl-N-nitrosourea, 1 gm ISOPAC container (Sigma N3385)
100% Ethanol

Phosphate/Citrate buffer: 0.1M NaH₂PO₄
0.05M Na₃C₆H₅O₇
pH to 5.0
Inactivating Solution: 20% w/v Na₂S₂O₃ in 0.1N NaOH
Disposable plastic cuvettes
10 and 30 cc syringes
18 gauge needle
1cc tuberculin syringes with 25 gauge, 5/8 inch needles
male mice 8-12 weeks of age

Stock Solutions:

1M Sodium Citrate:                              147 gm Na₃C₆H₅O₇
                                                             up to 500 ml dH₂O
1M Sodium Phosphate, monobasic       60 gm NaH₂PO₄
                                                             up to 500 ml with dH₂0

Working Solutions:

Citrate/Phosphate Buffer:        5.0 ml 1M Sodium Citrate (final concentration 0.05 M)
                                                10 ml 1M Sodium Phosphate (final concentration 0.1M)
                                                85 ml dH₂O
                                                pH to 5.0 with Phosphoric Acid
                                                0.45 μ filter
                                                Note: Citrate/Phosphate buffer is approximately 6.0 before
                                                PHing.

Inactivation Solution:              50 gm Na₂S₂0₃ (Sodium Thiosulfate)
                                                1 gm NaOH
                                                up to 250 mls with dH₂0

ENU Preparation

Note: Gloves and a lab coat should be worn at all times when working with ENU.

1) Prepare the phosphate/citrate buffer. Adjust the pH to 5.0 with phosphoric acid and 0.45 μ filter sterilize.

2) Working in a chemical hood inject 5.0 mls of 100% ETOH into the ISOPAC container. Gently agitate the suspension until the ENU goes into solution.

3) Inject 95 mls of phosphate/citrate buffer into the ISOPAC container, vented with an 18 gauge needle. Mix well.

4) ENU may be prepared fresh each time. Alternatively, it may be aliquoted into 15 ml polypropylene conical tubes and frozen at -80⁰C. Repeated freeze thawing should be avoided. The concentration of ENU should always be determined spectrophotometricly before use.

Spectrophotometric Determination of ENU Concentration:

1) Dilute 100 μl of the ENU suspension into 900 μl of phosphate/citrate buffer (1:10 dilution) in a disposable plastic cuvette.

2) Determine the OD₃₉₅ relative to a blank of phosphate/citrate buffer containing a 1:200 dilution of 100% ethanol.

3) Calculate the concentration of the ENU in solution given that a 1.0 mg/ml solution gives an OD₃₉₅ of approximately 1.0. The ISOPAC containers of ENU do not contain accurate weight amounts. The OD value is used to determine the exact concentration of your solution.

Note: ENU is light and pH sensitive. When possible keep vials covered with aluminum foil. Inject mice within 3 hours of preparing or thawing ENU.

Injection of Mice:

1) Weigh each mouse prior to injection.

2) Calculate, in mg/kg of body weight, the appropriate amount of ENU to be injected for each mouse depending on the desired dosage. Typically fractionated weekly doses are given at the same time each week.

3) In a chemical hood measure the appropriate volume of ENU into a 1 cc tuberculin syringe.

4) In a clean procedure room, wearing gloves, gown, and mask or under a chemical hood, inject the ENU intraperitoneally into each mouse. Injection volumes are typically between 0.2-0.4 mls per mouse. Animals may appear wobbly or lethargic after injection due to the alcohol content in the ENU.

5) Keep mice in a designated area and change bedding 24 hours after injection into a plastic bag containing paper saturated with inactivating solution.

Disposal of ENU:

1) Clean all spills and soak all equipment, syringes and gloves coming into contact with ENU with the inactivating solution and discard in waste.

2) Inactivate any remaining ENU with 20-30 mls of inactivating solution. Let stand in the chemical hood exposed to light overnight and discard.

The Jackson Laboratory