EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 548

for transcriptional regulation. The 5 -region upstream of the transcription start site
in the transporter gene is PCR-amplified using genomic DNAs and high-fidelity
DNA polymerase. The upstream and downstream primers are designed to include
internal restriction enzyme sites, since the PCR product is ligated into a firefly lu-
ciferase reporter gene vector such as pGL3-Basic (Promega Co.). After subcloning,
the 5 -deleted constructs are generated by digestion of the construct harboring the
PCR product with appropriate restriction enzymes and self-ligation. After cloning
of the deletion constructs, the nucleotide sequences are confirmed with a DNA
sequencer.
Cultured cells are maintained in the appropriate culture medium supplemented with
10% fetal calf serum and other appropriate additives. Cells are plated into 24-well
plates, followed by transfection of the reporter constructs and the Renilla reniformis
vector pRL-TK (Promega Co.) on the following day. Six hours after transfection,
the culture medium is changed to medium containing 2.5% fetal calf serum and
5% horse serum, and the cells are incubated for 48 hours at 37 ◦ C. The firefly and
Renilla lucuferase activities are determined 48 hours after the transfection using a
dual luciferase assay kit and a luminometer. The firefly activity is normalized to the
Renilla activity, and changes in the ratio of firefly to Renilla luciferase activities can
be used to identify the minimal promoter region. 59 , 60
In addition to determination of the minimal promoter region, computational se-
quence analysis can reveal transcription factor recognition sites, which may contribute
to the transcriptional regulation. Computational sequence analysis can be performed
with TFSEARC, TRANSFAC, or similar software.

Gel Mobility Shift Assay To confirm the possible contribution of transcription factor
recognition sites, gel shift assays are often performed to identify nuclear proteins that
interact with the sites. By using cell-derived nuclear extracts and a putative bind-
ing motif oligonucleotide within the promoter, protein-DNA interactions can be de-
tected. Confirmation can be obtained by using transcription factor-specific antibodies,
oligonucleotide competitors, or an excess of unlabeled probe oligonucleotide. 61
In the preparation of probes, oligonucleotides of the sense and antisense strands
containing putative binding sites of target nucleotide-binding protein are synthesized
and labeled with -[ 32 P]ATP. Commercial gel shift assays are available and are easy to

use. The binding mixture consists of 1 g of nuclear extract of cell, unlabeled competi-

tor probes, and several required components in buffer solution. After preincubation at
room temperature for 10 minutes, labeled probes are added, and the binding mixture
is incubated for a further 20 minutes. For supershift assays, 1

g of antibody is added
to a target protein 10 minutes before addition of the labeled probes. The volume of
the binding mixture is 10 L throughout the experiment. The DNA-protein complex

is then separated on a 4% polyacrylamide gel, and the gels are dried, followed by
autoradiography. 62
After the confirmation of transcription factor recognition sites, it is necessary to
investigate the effects of the transcription factor(s). For this purpose, luciferase as-
say with cultured cells expressing the transcription factor is convenient. In the same