ENU Mutagenesis of Toxoplasma

(adapted from Felice Kelly)

Use special caution, such as double gloves and fitted safety goggles, when working with highly mutagenic compounds and liquids. Collect solid and liquid waste separately for proper disposal. Work with ENU primarily in the chemical hood.

Decontamination solution (make fresh):

  • 10% sodium thiosulfate or 0.1 M KOH

Mutagenesis protocol

  1. Inoculate T25 tissue culture flasks confluent with human foreskin fibroblast (HFF) cells with 1 ml of freshly lysed tachyzoites and grow 18-25 hr at 37 °C under 5% CO2 in normal media. You will need as many T25s as you have mutagenesis conditions.
  2. Add DMSO to isopack of ENU (Sigma N3385-1G) to achieve 1 M. This is ~8.5 mL. Store 1 M stocks of ENU in the non-defrosting freezer in N200 in the marked red box.
  3. Add ENU (1 M Stock in DMSO) at a final concentration of 500 μM, 700 μM, and 1 mM. Add DMSO to 8 μl for each flask. Incubate for 2 hr in a 37°C incubator. Tightly cap flasks to avoid any accidental spilling of the mutagen.
    For T25s w/ 4 mL media this is:
    500 μM: 2 μL ENU
    700 μM: 2.8 μL ENU
    1 mM: 4 μL ENU
    2 mM: 8 μL ENU
    DMSO: 8 μL DMSO
  4. Pipette up and discard spent media into decontamination solution.
  5. Wash four times for 10 seconds at room temperature by rinsing the monolayer with 10 ml cold PBS (pre-cooled at 4 °C). All of this waste PBS and spent media needs to go into a decontamination solution, then the ENU waste. After these washes the parasites are considered non-mutagenic.
  6. Add 5 ml PBS, scrape and syringe lyse.
  7. Count the parasite concentration using a hemocytometer. Let the parasites settle for 5 min in the hemocytomer before counting.
  8. Pass most of the mutated population to expand.
  9. Inoculate 24-well plates w/50 parasites per well, triplicates for each condition.
  10. Wait 4 days.
  11. Aspirate medium, fix 5 min with 300 μL/well 100% methanol, stain with ~300 μL/well crystal violet solution for 30 sec, rinse with water and air dry. All at room temperature.
  12. Count plaques and select concentration of mutagen needed to achieve survival of 65% of the exposed parasites.
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