X. tropicalis ENU Mutagenesis
( Note: procedures for handling ENU, treated animals and waste were approved by the University of Virginia Office of Environmental Health and Safety and the Animal Care and Use Committee.)
One week prior to mutagenesis:
- Carefully select healthy male X. tropicalis frogs from the most inbred line currently available. At least 10 males should be used for each experimental variable (mutagen dose, husbandry condition, etc.).
- Set up tanks for mutagenized frogs close to the area where mutagenesis will actually be performed. Use a single tank for each experimental variable to be tested. Alternatively, PIT-tag or otherwise mark all animals so that individuals or groups can be readily identified.
- Animals should be placed in the tanks at least one week prior to the beginning of mutagenesis to allow time for acclimation to their new surroundings.
- Care for mutagenized animals should be carried out following the “Care for Mutagenized Animals” protocol.
- One week prior to mutagenesis, the following reagents should be ordered or made up:
- ENU (N-nitroso-N-ethylurea): order 1 gram “isopacs”, Sigma catalog # N3385. Order one “isopac” for each round of injections, store at -20 ° C until use.
- 1x PBS (phosphate-buffered saline), pH 6.0, autoclaved
Day of mutagenesis – all procedures should be done in a ventilated chemical hood and proper protective clothing should be worn (gloves, lab coat, safety glasses).
- Make up 1 liter of a 1/2000 dilution of MS-222 in 0.1x MBS (Modified Barth's saline), pH to 7.0 and keep at room temperature.
- Dissolve ENU in 1x PBS, pH 6.0. Pierce the rubber septum of the “isopac” with a 26-gauge needle to vent. Use a 50cc syringe with a second 26-gauge needle to inject 2x50ml PBS. After adding 100ml PBS to the “isopac”, remove both needles and shake vigorously for 5 minutes. ENU will not dissolve completely, but only one or two small clumps should remain.
a. NOTE: it is easier to remove the plunger and backfill the syringe twice than it is to draw 100ml of PBS through a 26-gauge needle).
b. NOTE: ENU is unstable and has a half-life of about 90 minutes at pH 6! ENU should only be dissolved immediately before use.
- Remove 5ml of ENU solution and transfer to a 15ml polypropylene tube. From this, draw up 0.09ml/frog in an insulin syringe (use the type with a needle guard at the end to prevent accidentally sticking yourself). Plan to inject 2 frogs per syringe.
- Anesthetize frogs. Place in MS-222 for 5-10 minutes until they become sluggish and do not thrash around when picked up. Do not let the frogs become heavily anesthetized (>10 minutes).
- Inject frogs one at a time. Wrap in a wet paper towel and expose the dorsal skin. Inject 0.1ml per animal into the dorsal lymph sac. Use exactly the same technique as when injecting HCG to stimulate egg-laying. Leave the needle in place for 2 seconds after injection, then slowly withdraw it. Be careful that no ENU flows back out of the hole.
- Place injected frogs in a moist chamber lined with water-saturated paper towels.
- After all injections are completed, take the moist chamber out of the chemical hood and place it on the bench next to the housing tanks. Regularly spritz the frogs with frog water to keep their skin moist.
- When animals are fully conscious (about one hour after injection), return them to their housing tanks.
- Dispose of all syringes as ENU-waste (sharps), dispose of bench paper and remaining ENU in “isopac” in ENU solid waste container. UVA Health and Safety says the amount of ENU remaining in the “isopac” can be disposed of as solid waste.
Repeat ENU injections at approximately one week intervals.