Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Gene Expression in Transfected Cells 357
below. This technique is equally applicable to adherent cells, but requires that
the cells are in suspension during the procedure. A number of alternative protocols
have been developed to allow electroporation of adherent cells in their
attached state (9–11).
2. Materials
2.1. Electroporation
1. Basic cell-culture equipment: 37°C carbon dioxide incubator and cell-culture hood.
2. Cell-culture medium (as used to culture your cells).
3. 25-cm2 (50 mL) cell-culture flasks.
4. Sterile 1.5-mL and 50-mL tubes.
5. DNA constructs (see Note 1):
a. Expression plasmid with your cDNA inserted downstream of a desired promoter.
The parental “empty” plasmid, lacking the cDNA, is needed as a control
to ensure that any phenotype seen is caused by the expression of the
transfected cDNA.
b. Internal control plasmid for the normalization of transfections, for example a
β-galactosidase gene under the control of a constitutively active promoter.
c. Luciferase reporter plasmid containing a luciferase gene under the control of
the inserted DNA-regulatory element whose activity you want to study.
Mammalian expression plasmids with different promoters and enzyme reporter
genes are available from a number of commercial sources, such as CLONTECH,
Invitrogen, Promega, and Stratagene.
6. Sterile electroporation cuvets with an electrode gap of 0.4 cm. These are available
from a number of companies, including Bio-Rad, Invitrogen, and Life Technologies
(see Note 2).
7. Electroporation system (pulse generator). These are available from a number of
companies, a popular model being the Gene Pulser ® II Electroporation System
(Bio-Rad).
8. Sterile Pasteur pipets.
2.2. Lysis of the Cells
1. 1.5-mL and 15-mL tubes.
2. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO 4,
1.8 mM KH 2PO 4. Adjust the pH to 7.4 with HCl. Store at room temperature.
3. Lysis buffer that permits β-galactosidase and luciferase assays (for example,
Reporter Lysis Buffer, Promega).
2.3. βββββ-Galactosidase and Luciferase Assays
1. 1.5-mL tubes.
2. β-galactosidase assay buffer: 60 mM Na 2HPO 4, 40 mM NaH 2PO 4, 10 mM KCl,
1 mM MgSO 4, 50 mM β-mercaptoethanol. Adjust the pH to 8.0 with NaOH. The
buffer can be stored at room temperature for several months.
3. ONPG (o-nitrophenyl-β-D-galactopyranoside) solution. Prepare prior to use by
dissolving in β-galactosidase assay buffer to a concentration of 0.8 mg/mL.