Biology Reference
In-Depth Information
10
6
cells/T
25
flask. EF cells can be
plated before or at the same time as the ES cells, and more EF cells can be plated
together to increase cell density and optimize growth.
them in ES medium in order to seed ~2-5
×
2.
Culturing ES cells: Grow cells in ES medium and split them ~1:6 (numbers refer
to the total plating area; 1 T
25
into 2 T
75
flasks) at ~70% confluency (i.e., 1-3 d
depending on the growth characteristics of the ES cell line). Change the medium
~1 h before splitting the cells. Wash the cells twice with PBS before adding
~0.3 mL/T
25
flask of trypsin. Make sure that the entire surface is covered and
incubate for 3-5'. Neutralize trypsin with at least 2 vol of medium. Dissociate the
cells by vigorous pipeting and transfer the suspension into a new flask. Although
complete removal of trypsin is not critical, one should try to dilute it at least 20
times (
see
Note 2
).
3.
Storage of ES cells: Trypsinize and pellet the cells, and then resuspend in freez-
ing solution at a density of ~10
7
/mL. Divide cells into 0.5 mL aliquots in
cryotubes and freeze. In contrast to thawing, ES cells should be frozen as slowly
as possible. Accordingly, place the cryotubes overnight at -80
°
C first and then in
a liquid nitrogen container for long-term storage.
3.2. Electroporation of ES Cells
1.
Prepare DNA by double CsCl-ethidium bromide centrifugation being sure to
remove all traces of salt and DNA-intercalating dye. Linearize the targeting vec-
tor at a restriction site placed at the junction between the plasmid and the insert
(if only G418 selection is used), or at the junction between the plasmid and the 3'
end of the TK-cassette (if double selection is used). DNA is ethanol precipitated,
washed, and resuspended at ~1
µ
g/
µ
L in TE buffer or deionized water
(7)
.
2.
Trypsinize rapidly growing ES cells making sure they are completely dissoci-
ated. Dilute an aliquot of the suspension ~10-fold and count the cells with a stan-
dard counting chamber.
3.
Wash the cells twice with 5 mL of electroporation buffer equilibrated to
room temperature.
4.
Transfer 50
L of linearized DNA in a sterile electroporation cuvette and resus-
pend the cells in the electroporation buffer to a final concentration of ~ 2
µ
10
7
cells/mL, making sure they are completely dissociated. Transfer 0.8 mL of the
suspension to the electroporation cuvette and mix with the DNA by pipeting the
solution several times without creating air bubbles. Put the cuvette in the electro-
porator, set a single pulse at 400 V-25
×
µ
F and leave it for 10' at room temperature.
5.
Transfer the content of the electroporation cuvette to a tube containing 10 mL of
ES medium, mix gently, but thoroughly, and plate cells on 100-mm plates which
have been gelatin-coated and covered with the EF layer ~12 h in advance. Start
selection 12-18 h later in G418-supplemented ES medium. If a positive-nega-
tive selection strategy is employed, gancyclovir should be also added at a final
concentration of 1.0
M
; in this case, a control plate with only G418 should be
used to monitor the efficiency of gancyclovir selection. After 7-12 d of selection,
pick singly or doubly resistant colonies.
µ
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