Biomedical Engineering Reference
In-Depth Information
8 As soon as colonies are clearly visible, trypsinize and plate half of the cells in a new
well of a 6 cm dish containing MEF cells with 5 ml ES medium
+
G418. Freeze down the
other half in ES medium (without G418) plus 10% DMSO.
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9 After the first trypsinization, the cells will grow faster. When cells in the 6 cm dish are
ready to be passaged, trypsinize and plate half the cells in a 10 cm dish with MEF cells
and freeze the other half as previously described.
10 When colonies have appeared in the 10 cm dish, passage to another 10 cm plate and
freeze approximately five vials, depending on colony density.
11 If necessary, propagate to another 10 cm dish for further expansion.
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Notes
cc
Once a positive clone has been identified by PCR, the appropriately labeled cells at
−
80
◦
C
should be expanded. To maximize ES cell viability, PCR screening should be completed within
one to two weeks.
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It will take several days before colonies grow up. They are often very sparse.
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For each round of freezing, the cryotube should be clearly labeled with the passage number.
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In addition, cells may be passaged to an uncoated plate in order to expand the cells
for genomic DNA extraction and subsequent Southern blot analysis to check for [1] single
integration of the targeting vector and [2] no unwanted genomic rearrangements.
PROTOCOL 10.19 Preparing Positive ES Cells
for Blastocyst Injection
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
•
10ml glass pipettes
•
50ml conical tubes
•
15ml conical tubes
•
1000
×
penicillin/gentamicin (0.59% penicillin (w/v)/8% gentamicin (w/v))
•
FBS (heat inactivated)
•
0.25% trypsin with EDTA
•
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 (50 mg/ml)
