Biology Reference
In-Depth Information
6.
With scissors separate, each implantation site from the other. With fine forceps,
carefully free embryos from the uterine wall, yolk sac, and placenta. Repeatedly
wash them in several Petri dishes filled with fresh sterile PBS, until bleeding stops.
7.
With fine scissors, cut and discard the head. Open the abdomen with fine forceps
and remove all internal organs.
8.
Hold the carcass with forceps above a Falcon tube filled with trypsin (1 mL/
embryo) and mince it with fine scissors to very small pieces that are let to fall
into the trypsin solution.
9.
Incubate the suspension at 37
C for 15 min. Break tissue pieces by pipeting up
and down with a large gage pipet (e.g., 10-mL pipet). Incubate again at 37
°
C for
15 min. Thoroughly, dissociate cell clumps by pipeting up and down with a small
gage pipet (e.g., a 2 mL or a Pasteur pipet).
°
10.
Fill the falcon tube with feeder medium and let it stand for 5 min to let large
aggregates sink. Transfer the supernatant to a fresh falcon tube and centrifuge it
at 120
g
for 5 min. Discard the supernatant and dissolve the pellet with 2 mL/
embryo of feeder medium. Seed 1 mL into a 15-cm cell-culture Petri dishes filled
with 14 mL of feeder medium (2 Petri dishes/embryo).
11.
Grow to confluency without changing medium in a 37
C, 5% CO
2
incubator.
Wait for other 3 d. Wash plates with 15 mL PBS and incubate for 5 min inside the
incubator with 2 mL of trypsin. Resuspend detached cells with 2 mL of feeder
medium. Reseed the plate with 0.5 mL of cell suspension.
°
12.
Pellet cells in a Falcon tube. Discard supernatant and irradiate the cell pellet with
6000 rad to inhibit cell division.
13.
Resuspend the irradiated pellet with freezing medium (3 mL/Petri dish). Aliquot
1 mL of cell suspension/cryovial and freeze in a box kept in frozen carbon diox-
ide. Keep frozen stocks either at -80
°
C or in liquid nitrogen.
14.
Add 14 mL of feeder medium to reseeded cells and repeat from
step 11
for a
maximum of two times.
15.
To check for sterility, thaw frozen aliquots at 37
C. Add cells to 5 mL of feeder
medium in a 10-mL sterile tube. Centrifuge 5 min at 120
g
. Decant supernatant,
resuspend pellet and seed it in a 10-cm cell-culture Petri dish. Culture cells for
few days and then test supernatant for mycoplasma infection.
°
Frozen feeder fibroblasts can be thawed and plated in advance or just
together with ES cells (in the presence of ES medium). To obtain a confluent
layer of irradiated cells, thawing one frozen vial normally gives enough cells to
cover the surface of a 10-cm diameter Petri dish. All other areas should be
calculated using that rule of thumb.
3.3.2. Electroporation and Selection
Before transfection, ES cells must be expanded and kept growing minimiz-
ing passages. The construct is transfected into ES cells by electroporation. This
method assures that in most cases only one copy of the exogenous DNA is
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