Biomedical Engineering Reference
In-Depth Information
•
1
×
Dulbecco's PBS
•
10ml glass pipettes
•
15ml conical tubes
•
1000
×
penicillin/gentamicin (0.59% penicillin (w/v)
/
8% gentamicin (w/v))
•
DMEM (high glucose, high bicarbonate, without pyruvate, with
L
-glutamine,
Invitrogen)
•
FBS (heat-inactivated, Hyclone)
•
MEF medium: DMEM, 1
×
penicillin/gentamicin, 10% FBS
•
0.1% gelatin-coated 10 cm Petri dishes.
Method
1 Remove a cryotube of
γ
-irradiated MEF cells from liquid nitrogen and warm it in a 37
◦
C
water bath.
2 Transfer cells to a 15 ml conical tube with 7 ml of MEF medium.
3 Spin cells at 600
g
for 5min.
4 Resuspend cells in 9 ml of MEF medium.
i
5 Distribute cells to several 10 cm gelatin-coated plates.
6 Add MEF medium to make up a total volume of 10ml per plate.
j
,
k
7 Each plate will be ready to receive ES cells after 24-48 h.
Notes
i
Usually 1.5 million cells will be sufficient to plate one 10 cm plate.
j
MEF cells will adhere rapidly so sometimes it is better to have the 10 cm plates ready with
several ml of medium.
k
It is important to make sure that the MEF cells are fully spread out over the plate, with no
large gaps. This ensures a sufficient adherence substrate for the ES cells.
10.2.4.2 ES cell culture protocols
Once the targeting vector has been constructed and MEF feeder cells are prepared, genetic
manipulations of ES cells in culture may proceed. An overview of the steps involved in
achieving successful gene targeting in ES cells is found in Figure 10.3. These steps involve
growth of ES cells, transfection of ES cells with targeting vector via electroporation, picking
of colonies obtained after positive-negative selection, harvesting of colonies and screening
of ES cell DNA by PCR (see Protocols 10.11-10.19). It is imperative to plan ahead metic-
ulously, as feeder cells must be plated at least 48 h before plating, passaging or picking
ES cells.
