Elucidating Gene Function through Use of Genetically Engineered Mice - Genomics: Essential Methods - page 227

Targeting vector
construction

ES cell
culture & passage

ES cell
electroporation

ES cell
colony picking

ES cell
harvesting

PCR screening
of ES cell DNA

~7 days 3-5 days

~7 days

ES cell
storage at -80

° C

Figure 10.3 Outline of ES cell manipulations for gene targeting. MEF feeder cells must be plated
2 days prior to ES cell plating or passage. Arrows depict approximate time from one step to the
next (shaded boxes). ES cell colony picking usually lasts for 5 days; harvesting occurs 3-5 days
from the time of individual colony picking. Harvested colonies are screened immediately by PCR
for homologous recombination.

PROTOCOL 10.11 Plating ES Cells

Equipment and reagents
• Incubator at 37 ◦ Cwith5%CO 2
• Water bath at 37 ◦ C
• 10ml glass pipettes
• 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)
• β -Mercaptoethanol (BME)

ESGRO leukemia inhibitory factor (LIF; 10 7 units/ml, Chemicon, cat. no. ESG1107)

•

ES medium: DMEM, 20% FBS, 1 : 1000 dilution of penicillin/gentamicin, 1 : 100 dilution
of BME, 1 : 10000 dilution of LIF

•

γ -irradiated MEF cells plated on 0.1% gelatin-coated 10 cm plates 48 h prior to ES cell
plating.

•

Method

1 Pre-warm ES medium in a 37 ◦ C water bath.
2 Aspirate MEF medium from one 0.1% gelatin-coated 10 cm dish previously plated with
γ -MEF cells, and add 7 ml of ES medium to the MEF cells.