Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Gene Expression in Transfected Cells 361
3. The optimal amount of expression plasmid depends on the cell type and the efficiency
of the transfection and the expression of the plasmid in these cells. It might
also depend on effects of the introduced plasmid or the expressed protein, for
example if the protein becomes toxic to the cell above a certain concentration.
Therefore try a range of DNA concentrations to determine the optimal concentration
for your purposes.
4. The cells can alternatively be resuspended in PBS or PBS supplemented with
20 mM HEPES, pH 7.1, for the electroporation procedure. HEPES provides extra
buffering capacity to reduce the pH change that occurs at the electrodes, a cause
of cell death during electroporation. There appears to be little difference in transfection
efficiency between PBS, PBS-HEPES, or culture medium, but cells may
show enhanced survival when electroporated in culture medium. Transfection
efficiency can be affected by the salt concentration of the electroporation buffer
(12). Furthermore, addition of carrier DNA can improve transfection efficiency
(12). It is therefore recommended to use carrier DNA, such as an inert plasmid,
when the amount of expression plasmid is low.
5. For cells more resistant to transfection, efficiency may be improved by incubating
the cells (in the cuvets) on ice for 5 min before and after electroporation. This
may reduce the kinetics of pore closure and thus provide more time for the DNA
to enter the cell. It may also protect the cells from heat damage when subjected to
the electric pulse.
6. The voltage setting is the most critical parameter of the transfection procedure.
Too low a voltage will have no effect on the cell membrane, but too high a voltage
irreversibly damages the cell. The optimal voltage should be determined empirically
using your experimental conditions. As a guideline, Table 1 lists voltages
that are successful for a variety of cell lines using the indicated electroporation
system. Comprehensive studies of optimal voltages for other cell types appear in
the literature (for example, refs. 4,12–14).
7. Transfected cells can be analyzed several hours to several days after
electroporation. Times shorter than 8 h are usually not long enough for reasonable
expression of the protein. Over a period of several days, the cells lose transfected
DNA and thus expression from the plasmid will gradually decrease. For
studies of expression over a longer period of time, stable transfectants are needed.
Albeit at a very low frequency, exogenously introduced DNA can insert into the
chromosomal DNA. If the transfected expression plasmid also encodes a selectable
marker, for example a drug resistance gene, it is possible to select for and
amplify these cells (15–17). These stable transfectants can continue to express
the integrated gene for an indefinite time.
8. If the β-galactosidase activity of the samples varies more than fourfold, it is not
possible to stop the reactions when all samples lie in the OD 420 range of 0.2–0.8.
In this case, stop individual reactions when they have reached the appropriate
color, record the reaction time, and wait until all reactions are finished before
measuring the OD 420 of the samples. The ONPG hydrolysis per time unit is then
calculated.