Mouse Mutagenesis with ENU

Protocol Index

 

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 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 -80°C. 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 OD395relative 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 sensitive 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 in 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.

Equipment and Reagents:

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

    1 cc 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 dH2O


    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 before pHing will be approximately 6.0.

    Inactivation Solution:

  • 50 gm Na2S2O3 (Sodium Thiosulfate)
  • 1 gm NaOH
  • up to 250 mls with dH2O

    Protocol Index

     

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    Program for Genomic Applications (PGA)

    Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)