Biology Reference
In-Depth Information
3.8.2. Induction of ES Cells-Derived Teratomas
1.
Grow ES cells with standard methods to obtain at least 10
8
cells.
2.
Wash cells two times with PBS and incubate with trypsin for 5 min at 37
C. Add
ES medium and disperse aggregates by gently pipeting cells up and down. Add
ES medium if needed and let feeder cells attach to the culture dish for 30 min in
the incubator.
°
3.
Gently wash the plate to collect nonadherent ES cells and save the supernatant in
a sterile tube. Count the cells with a Burcker's chamber.
4.
Centrifuge 10
7
cells 5 min at 120
g
. Resuspend the pellet in PBS.
5.
Spin cells down and resuspend the pellet in 300
µ
L. Load the cell suspension in a
1-mL syringe.
6.
Anesthetize a 129 male mouse and inject cells subcutaneously.
7.
The tumor should be clearly visible in 15-20 d. When it has reached the desired
dimension, excise it and treat it for histological analysis. As with embryoid bod-
ies, teratomas can be collected in sterile conditions and trypsinized to culture
differentiated cells.
4. Notes
1.
Stringency varies depending on the homology of the probe with mouse DNA. In
case the probe shows a homology from 80% to 100% hybridization can be car-
ried out at 65
°
C and followed by two washes of 30 min at 65
°
C with 2X SSC,
1%SDS, and 0.4X SSC, 1%SDS.
2.
Southern blot analysis of mutant ES cell clones is preferred over PCR analysis.
Beside the fact that homologous recombination should always be confirmed by
Southern blot, this method is more reliable than PCR because it essentially gives
no false positive/negative signals.
3.
Flanking arms can be asymmetrical, but better results have been obtained when
the shorter arm is not smaller than 2 kb.
4.
A viable homozygous knock-out mouse is often suitable for such purpose. The
expression of a resistance gene (usually the
neo
r
gene) is guaranteed by the fact
that the mutant ES cells from which the mouse line has been derived, had been
selected in a similar way.
5.
Plugs are unstable and easily lost during the day that follows mating. It is there-
fore important to check for plugs early morning and not later than 11
AM
. To ease
inspection, it is useful to use a blunt, sterile probe such as a flame-sealed tip of a
Pasteur pipet.
6.
In commercial preparations of G418, only a fraction of the total weight is the real
active compound. The routine use of high concentration (400
g/mL) of crude
powder has proven to be adequate to avoid testing of different antibiotic batches.
µ
7.
Colonies should be picked as soon they start to be detectable by eye inspection.
Choosing the picking time is a critical step: whereas waiting too long can result
in differentiation of colonies, retrieval of small aggregates supplies too few cells
for the subsequent expansion.
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