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Transgenic Resources Program
Resource Services and Contacts | Staff | TRP
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EMBRYONIC STEM CELL TRANSFECTION
Construct Preparation
Prices and Guarantees
Embryonic stem (ES) cells are grown on primary mouse embryo
fibroblast feeders (PMEF) in the presence of leukemia inhibitory
factor (LIF). Approximately 2 x 107 ES cells are mixed
with 30-50 µg of linearized construct prior to electroporation.
Twenty-four hours after electroporation, ES cells
are exposed to antibiotic selection, usually 180 µg of G418. Resistant
colonies appear on the plates 10-12 days after transfection.
Generally, up to 96 clones are picked and transferred
to individual wells of 24-well tissue culture plates
and grown up for DNA analysis. Duplicate 24-well plates
are made from the same set of resistant clones and
frozen. When clones carrying a homologous targeting
event have been identified from the first plate, those
clones on the stored plate will be thawed and expanded
for a bulk freeze and to provide 1,10cm plate per clone
for Southern analysis. After the targeting events are
confirmed by Southern analysis,
these cells will be thawed and used for microinjection.
Note: A common mistake made by first-time investigators is to
begin transfection before choosing a proper protocol
for determining targeted events. Once the 24-well plates
are frozen, thawing and expanding these cells to generate
more material for analysis is unwise and generally unsuccessful.
The process would involve passaging an unwieldy number
of clones, and freezing another set of 24-well plates. Therefore, it
is highly recommended that a positive control vector be
made if analysis is by PCR. The positive control is composed
of the targeting construct extended to include the fragment
encompassing the intended primer sequence outside the
area of homology. Thus,
the primer set would reveal random integration events
of the positive control. Positive
controls will be electroporated at no charge to test the
analysis scheme on ten resulting G418 resistant clones. If a positive
control is not available and no correctly targeted clones
are identified following the initial electroporation, a fee
will be incurred for the resulting excess tech time and reagents
needed to help troubleshoot the analysis.
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CONSTRUCT PREPARATION
Each electroporation
event uses 30-50 µg of linearized
construct. The more DNA available for the electroporation
mixture, the more likely it will integrate within
the DNA in the host cell. The construct should
be at a concentration of 1 µg/ul and free of contamination
from ethidium bromide, ethanol,
phenol, chloroform, and high salt. Constructs
may be delivered to the Health Sciences Building,
room T-140 during normal working hours. Ask for Sylvia Roh (slroh@u.washington.edu) or Bob Hunter.
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ES CELL TRANSFECTION PRICES
AND GUARANTEES
The charge includes ES cell transfection (20 million
cells), selection, clonalisolation, plates for
storage at -80° C,
duplicate plates for analysis, thaw and expansion of targeted clones
for freeze at -196° C and one 10cm plate per
targeted clone for Southern Analysis. Because different selection vectors included
within the targeting construct result in widely variable enrichment
for targeted events, no guarantees as to the number of clones arising
from electroporation can be made. However, if 50% or more of
the successfully targeted clones are lost through
the freezing process the transfection process will be repeated at no
additional cost.
Click here for rates.
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