Mutagenesis of Embryonic Stem Cells with Ethylmethanesulfonate (EMS)
Material and Methods:
35 mm, 60 mm, 100 mm and 150 mm tissue culture treated dishes
ES media
Trypsin/EDTA (Gibco 25200-056)
PBS (Gibco 21600-069)
Gelatin (Sigma G2500)
2M Sodium Thiosulfate, Na2S2O3 (Sigma S1648)
Mitomycin C (Sigma M4287)
Methanesulfonic Acid Ethyl Ester, EMS (Sigma M0880)
MEF for use as feeders
2 Amino 6 Mercaptopurine or 6 thioguanine, 6TG (Sigma A4882)
DMSO for freezing cells (Sigma D2650)
ES cells
Solutions:
6 TG made to 10 mg/ml in 0.2 N NaOH - Aliquot and store at -20°C
Inactivation Solution: 79 gm Na2S2O3 (Sodium Thiosulfate) up to 250 mls with dH20
Based on Munroe et al (2000) Nature Genetics 24:318-321
Note: EMS is a mutagen and should be disposed of properly. All equipment, pipets, plates and gloves coming into contact with the EMS should be rinsed with 1.0 M Na2S2O3 before disposal. All liquids should be brought to a final concentration of 1.0 M Na2S2O3 for at least 10 minutes before disposal (De Meo et al 1990).
ES Cell Treatment:
1. Plate ES cells onto 35 mm dishes with feeders at a concentration of 2X106 cells/dish. Incubate at 37°C in 5% CO2 overnight. We currently use v6.4 (129/B6) F1 hybrid ES cells.
2. Change the medium the following morning with 2.0 ml of fresh ES cell media.
3. Wait at least 4 hours before adding EMS directly to each dish to a final concentration ranging from 0.4 mg/ml to 0.8 mg/ml. Higher concentrations of EMS have been shown to result in increased mutation rates.
4. Incubate 16-20 hours at 37°C in 5% CO2.
5. Wash plates 3 times with 2.0 mls of 1X PBS. Trypsinize and count the cells. Cells will be divided into three sets, one for cell death determination, one for expansion and freezing, and one for a hprt (hypoxanthine phosphoribosyltransferase) assay to determine mutation rate.
Note: Successful treatment of 129 ES cell lines CT129 and CJ7 with EMS has also been reported by Munroe et al. (2000), Nature Genetics 24:318-321, and successful treatment of CT129 ES cells with ENU has been reported by Chen et al. (2000), Nature Genetics 24:314-317.
Calculation of Cell Death:
Plate 1 X 103 EMS treated and untreated (control) cells onto two 60 mm gelatinized dishes each without feeders to determine the degree of cell death. Count the colonies after approximately one week. To control for plating efficiency and provide an estimate of cell death, colony counts will be compared to the untreated plate. The number of colonies from the EMS treated plate divided by the number of colonies from the untreated plate x 100 equals the percent survival. A survival range of 10-30% is typically associated with high efficiency mutagenesis.
Cell Expansion and Freezing:
Option 1: Plate Pool - Plate one half of the treated cells onto a 60 mm gelatinized dish with feeders. Pass as needed. Cells may be expanded further at this point or frozen before expansion. This population of ES cells will be used for injection of blastocysts to make chimeric mice. Using this approach, each chimera produced is likely to represent a different diploid mutagenized genome and may represent more than one diploid mutagenized genome. To date, our efforts have used a pooled plate of ES cells to produce germline competent chimeric mice.
Option 2: Clone
1. Plate one half of the cells onto 60 mm gelatinized dishes with feeders at three different dilutions, one heavy plate for freezing and the other two lighter for picking colonies. Remember that lower doses of EMS have higher cell survival rates and should be plated at a lower density than higher doses for picking colonies.
2. When cells are ready, pick colonies onto gelatinized 48 well dishes with feeders. Expand as desired and freeze. Each chimeric mouse produced from an isolated ES cell colony represents one diploid mutagenized genome.
Hprt Assay:
1. Plate the other half of the cells onto a 60 mm gelatinized dish without feeders. Cells must be passed for 9-14 days off feeders prior to treatment to deplete any feeder derived hprt activity.
2. After two weeks of passing the cells, trypsinize and count. Plate 2 X 106 cells onto five 150 mm gelatinized dishes without feeders. Add 6TG (thioguanine) to a final concentration of 10 μg /ml. Feed as needed with media containing 10 μg/ml of 6TG for 9 to 14 days. Count colonies after two weeks.
3. Also plate 1 X 103 cells onto two 60 mm gelatinized dishes without feeders to determine plating efficiency. Count the colonies after approximately one week. These cells will be used to determine the number of cells that survived plating.
4. To determine the mutation rate at the hprt locus, multiply the percentage of cell surviving plating by 2 X 106 cells plated and then divide by the average number of colonies on the hprt assay plates.
Note: Not all cell lines will tolerate 10 μg/ml of 6TG. The appropriate concentration has to be determined for each cell line.
