PXR, in the presence of a CYP3A4 inducer, binds to and activates the
responsive elements in a reporter gene construct, leading to higher expression
of a reporter gene, which is usually luciferase, chloramphenicol acetyl
transferase (CAT), or secretory placental alkaline phosphatase (SPAP). The
expression of the reporter gene is determined, and the ratio between the
treatment and vehicle control is used to estimate human PXR-dependent
induction potential of the test compound.
Variations of the PXR reporter gene assay have been developed in a
number of laboratories. In our laboratory, DNA vectors were constructed
FIGURE 17.2 Illustration of cell-based PXR reporter gene assay. HepG2 cells are
cotransfected with three vectors, pcDNA3–hPXR, pGL3–3A4, and pSEAP2. The
pcDNA3–hPXR codes for expression of human PXR, whereas pGL3–3A4 is a reporter
vector, in which the PXRE is inserted. A CYP3A4 inducer such as rifampin binds and
activates the expressed human PXR, which in turn binds to PXRE in the reporter gene
construct, and activates the transcription of reporter gene firefly luciferase. The
luciferase activity, determined by Steady-Gloluciferase assay, is proportional to the
activation of PXR by the inducer. pSEAP2 activation results in expression of alkaline
phosphatase activity, which is used to normalize the expression of luciferase activity.
(Goodwin et al., 1999; Luo et al., 2002).
ASSESSMENTS 559