Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

356 Hughes et al.
The electroporation procedure entails mixing cells with DNA and subjecting
them to a high-voltage electric field, which transiently permeabilises the
cell membrane allowing DNA to enter the cell (reviewed in refs. 4 and 5).
Once inside the cell, the DNA is transported to the nucleus and transcription is
initiated from the promoter of the expression plasmid. The choice of promoter
is important in that it should enable a high level of transcription of the cDNA in
the chosen cell type. Viruses such as the human cytomegalovirus (CMV), simian
virus 40 (SV40), and Rous sarcoma virus (RSV) have naturally evolved
strong promoters that function in a variety of cell types and are often the best
choice. The level of protein expression also depends on a number of other factors,
such as efficiency of transfection, cell type, and regulation of the protein
by post-transcriptional mechanisms. For example, calmodulin is efficiently
regulated post-transcriptionally (6–8), warning that profound increases in
mRNA levels by transfection will not necessarily result in correspondingly profound
increases in protein levels.
To monitor the success of a transfection, it is common to include an internal
control plasmid. In analogy to the expression construct, this plasmid contains
the gene for an enzyme cloned downstream of a constitutively active promoter.
At an appropriate time after transfection, a sample of cells is lysed and assayed
for enzyme activity to ensure that the DNA was successfully delivered into the
cells. By comparing the enzyme activity in the same number of cells from different
transfections, it is also possible to normalize and thereby compare different
transfections.
It is far beyond the scope of this chapter to discuss the many possible analyses
of functional consequences of (over-) expressing a calcium-binding protein
or a protein regulated by a calcium-binding protein in living cells. However,
one general process that can be readily assayed is the effect of the transfected
protein on transcription from a gene control region. The regulatory sequence of
interest is cloned into a plasmid upstream of a gene for a reporter enzyme. The
chosen enzyme should be absent from mammalian cells and its activity easily
measured. Luciferase is usually used for this purpose, as it can be measured by
a convenient and very sensitive assay, whereas a gene for another luciferase
enzyme that can be assayed independently, β-galactosidase or chloramphenicol
acetyl transferase (CAT) is used in the internal control plasmid. The amount
of reporter enzyme produced, determined by measuring its activity in a cell
lysate, is proportional to the amount of transcription initiated from the regulatory
sequence. The appeal of this simple method is that it also allows mutational
analysis to define regions of a regulatory element that are important for
the studied transcriptional regulation, as well as analyses of effects through
individual transcription factors by using isolated specific DNA binding sites.
A standard protocol for transient transfection of suspension cells by
electroporation and analysis of the expression of reporter genes is described