Modified from Lyle Zimmerman. Special thanks to Nick Hirsch, Mary Burns, and Rob Grainger for advice.

Note: ENU is a potent toxin, mutagen, carcinogen. Please take appropriate precautions to protect yourself and disposal of ENU. We highly recommend that you discuss this with your Environmental Health & Safety group to insure that you use ENU safely.

Sperm Mutagenesis

Prepare animals

day 0

prime 4 females (10 U hCG)

boost 4 males (100 u hCG)

day1

boost 4 females

sacrifice 4 males when females begin to lay.

ENU work:

Necessary Reagents

MES (10 g)

Sodium thiosulfate (500 g)

NaOH

prepare hood, floor around hood, waste containers

double gloves, wristguards, respirators, etc.

prepare decontamination bath (20% sodium thiosulfate 2% sodium hydroxide). This is a 2x bath and can be used 1:1 with decontaminated solutions.

Use a full 500 g sodium thiosulfate (just use a whole bottle) and 50 g NaOH into 2.5 L of solution.

All ENU-contaminated tips, tubes etc. go into the bath for 24 hours

prepare 100mM ENU stock:         

Make 50 ml 1 M MES

9.76 g MES into a final volume of 50 ml

Add 5 pellets of NaOH (helps get it into solution)

Add NaOH pellets or 10 M NaOH to final pH (7.0)

Make 5 mM MES pH 7.0 - 1 ml 1 M MES into 199 ml H₂0

inject 85.4 ml 5mm MES pH 7.0 (2-[N-Morpholino]ethanesulfonic acid, Sigma M-3671) into ENU (N-Nitroso-N-ethylurea Sigma N3385 1g isopac), rock rt until dissolved (up to 4 hrs, triturate carefully w/ 18gauge needle if necessary; last bits may refuse to go into solution); use fresh and freeze aliquots for later. Some workers recommend OD’ing the solution to accurately determine concentration.

Determining [ENU] by Spectrophotometry

Method:

1. Dissolve 1 gm Isopac ENU in 100ml ENU dilution buffer. This solution is approximately 10 mg/ml.

2. Dilute 20µl ENU solution to 1 ml with ENU dilution buffer (i.e. 1:50 dilution)

3. Determine OD₃₉₈ using a disposable plastic cuvette. Note: This is an important step. We and others have found that the amount of ENU varies substantially from lot to lot.

4. 1 mg/ml solution of ENU gives OD₃₉₈ =0.72.

            Therefore,             [ENU] mg/ml = (OD₃₉₈)(50)/0.72

            or                                                = (OD₃₉₈)(69.4)

We would expect ~ 11.7 mg/ml

Divide measured concentration by 11.7 to generate correction factor. Multiply correction factor by doses to ensure accuracy.

BSA- or serum-coat tubes, dishes, pipets that will contact sperm solution

Treat Sperm with ENU

Make L15+10%CS+3mM MES (pH 7) - 30 ml 1 M MES into 10 ml L15+10%CS –(need 90 ml L15 + 10 ml L15+10%CS)

dissect 8 trop testis (4 frogs) into L15+10%CS, place @ 4C.

Use a 1.5 ml eppendorf tube

Remove L15+10%CS and add 500 ml L15+10%CS 3mM MES pH 7

macerate testis

bring up to 1.8 ml w/L15+10%CS +3mm MES pH7.

Remove 0.1 ml and place aside – control for sperm fertility

Setup 15 ml conical tubes.

Transfer sperm solution to 15 ml conical tubes (see below)

Incubate at RT for 10 min mixing occasionally

wash 2x by adding 10ml L15, spinning 5' 1000 rpm clinical fuge

Resuspend pellet by gentle flicking of the tube and not pipetting.

Start squeezing frogs after second spin

resusp. sperm pellet in ~1ml L15+10%CS.

Fertilize Eggs

Squeeze eggs into dry L15+10%CS coated dish

Apply sperm and wait 10 min.

Flood with 1/9xMR

assay for cleavage two hours later (fertilizations can be delayed)

dejelly and sort into 1/20xMMR+gent

day2

sort viable embryos into 1/20xMMR+gent. Also sort and count samples of control and mutagenized embryos to assay dominant effects.

Inject Frogs

Male frogs typically weigh between 6-9 g (Weigh frogs in 1 qt Glad plastic containers – this also acts as recovery chamber)

Inject 100 mg/kg or 0.1 mg/g of ENU

1 g ENU is diluted in ~85.4 ml of Buffer

Therefore inject [frog wt (g)]*[0.1mg/g]*[1ml/11.7mg ENU] or 0.00855*frog wt.

This should be corrected based on actual measured amount of ENU determined by spectrophotometer

Volume injected should vary between 250-400 ml round to the nearest 50 ml. So dilute your ENU based on the ENU correction factor to make the injections between 250-400 ml.

So assuming that the heaviest frog is 10 g then

Dilute 1:[correction factor]*4.68

inject [frog wt (g)]*0.04 in ml