Elucidating Gene Function through Use of Genetically Engineered Mice - Genomics: Essential Methods - page 238

• 15ml conical tubes
• 1000 × penicillin/gentamicin (0.59% penicillin (w/v)/8% gentamicin (w/v))
• FBS (heat inactivated)
• DMEM (high glucose, high bicarbonate, no pyruvate, with L -glutamine, Invitrogen, cat.
no. 11965)
• BME
• ESGRO LIF (10 7 units/ml, Chemicon, cat. no. ESG1107)
• G418/geneticin-selective antibiotic (50mg/ml)
• FIAU (1 mg/ml, dissolved 1 : 1 in ethanol/H 2 O, = 2.7 m M for 10 000 × stock, Moravek
Biochemicals)
• MEF medium: DMEM, 1 : 1000 penicillin/gentamicin, 10% FBS
• ES medium: DMEM, 20% FBS, 1 : 1000 penicillin/gentamicin, 1 : 100 BME,
1 : 10 000 LIF
• ES medium + selection:
—ESmediumplus:
—1 × (1 : 10 000 dilution of) FIAU
—1 × (1 : 167 dilution of) G418 (e.g. 3 ml for 500ml of medium)
• γ -irradiated MEF cells plated on gelatinized plates 48 h prior to ES cell plating
• 1 × Dulbecco's PBS
• 0.25% trypsin with EDTA
• DMSO

Cryotubes. •

Method

1 Plate MEF cells on 0.1% gelatin-coated six-well dishes 24-48 h in advance. cc
2 Pre-warm ES medium to 37 ◦ C.
3 Fill a 15 ml conical tube with 9 ml of ES medium + 1 × G418.
4 Quickly thaw tubes with positive cells in a 37 ◦ C water bath.
5 After thawing, transfer each clone to a conical tube with 9ml of medium and spin for
3 min at 600 g .
6 While spinning down cells, replace MEF medium in the six-well dish with 2 ml of ES
medium + 1 × G418.
7 Resuspend the pelleted cells in 1 ml ES medium + 1 × G418 and plate onto one well of
a six-well dish. Change the medium daily. dd