A new model for studying tissue-specific mdr 1a gene expression in vivo by live imaging

Abstract

Multidrug resistance continues to be a major impediment to successful chemotherapy in cancer patients. One cause of multidrug resistance is enhanced expression of the mdr1 gene, but the precise factors and physiological conditions controlling mdr1 expression are not entirely known. To gain a better understanding of mdr1 transcriptional regulation, we created a unique mouse model that allows noninvasive bioimaging of mdr1 gene expression in vivo and in real time. The model uses a firefly luciferase ( fLUC) gene inserted by homologous recombination into the murine mdr1a genetic locus. The inserted fLUC gene is preceded by a neo expression cassette flanked by loxP sites, so that Cre-mediated recombination is required to configure the fLUC gene directly under the control of the endogenous mdr1a promoter. We now demonstrate that the mdr1a.fLUC knock-in is a faithful reporter for mdr1a expression in naive animals, in which fLUC mRNA levels and luminescence intensities accurately parallel endogenous mdr1a mRNA expression. We also demonstrate xenobiotic-inducible regulation of mdr1a.fLUC expression in real time, in parallel with endogenous mdr1a expression, resulting in a more detailed understanding of the kinetics of mdr1a gene induction. This mouse model demonstrates the feasibility of using bioimaging coupled with Cre/loxP conditional knock-in to monitor regulated gene expression in vivo. It represents a unique tool with which to study the magnitude and kinetics of mdr1a induction under a variety of physiologic, pharmacologic, genetic, and environmental conditions. bioimaging ͉ conditional knock-in ͉ gene regulation ͉ MDR1 ͉ multidrug resistance T he human MDR1 gene encodes P-glycoprotein (Pgp), which functions as a transmembrane drug transporter and mediates the efflux of drugs from cells, thus conferring multidrug resistance on cancer cells and tumors that over-express it (1, 2). In addition, basal expression of MDR1 in organs such as liver, kidney, and colon, because of their involvement in drug excretion and absorption, affects the pharmacokinetics of drug uptake and excretion for agents that are substrates for Pgp transport (3-6). The mechanism of MDR1 regulation in tumors or in normal, nonmalignant tissue, particularly in response to xenobiotics and other environmental and physiologic stimuli, is not fully understood. The tumor-suppressor p53 has been implicated as a possible transactivator of MDR1 expression in tumors (7-9), although this association remains controversial (see ref. 10). In addition, nuclear receptors such as SXR and constitutive androstane receptor (CAR) have been suggested as possible master regulators of xenobiotic-and druginducible expression of MDR1, and of other genes involved in drug metabolism in organs such as the liver, kidney, and colon (11,12). One recent report implicates the transcription factor FOXO3a in doxorubicin-mediated induction of MDR1 in the K562 human leukemia cell line (13).Our limited understanding of MDR1 gene regulation in vivo is partly a result of the difficulty in...

In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice

“…A mouse mdr1a-luc model has previously been generated in which the luciferase reporter was inserted into the genomic locus of the mouse mdr1a gene by homologous recombination (Gu et al, 2009(Gu et al, , 2013 and bioluminescent imaging was used to study in vivo transcription of the mouse mdr1a promoter. However, mdr1a transcription was not reported in the mdr1a-luc mouse CNS.…”

Molecular Imaging of Membrane Transporters’ Activity in Cancer: a Picture is Worth a Thousand Tubes

“…The first transgenic mouse reporter model was based on a firefly luciferase (fLUC) cDNA inserted into the murine mdr1a genetic locus by homologous recombination and allowed the imaging of the mdr1a gene expression (104). Using this model, it was demonstrated that abdominal luminescence intensity reflects basal and pregnenolone-16α-carbonitrile (PCN)-induced intestinal mdr1a and fLUC mRNA expression.…”

Bioimaging Real-Time PXR-Dependent mdr1a Gene Regulation in mdr1a.fLUC Reporter Mice

“…Matched vehicles were used as controls. In vivo imaging of abdominal luminescence was performed in mice heterozygous for the mdr1a.fLUC allele (mdr1a 1/fLUC ) as described previously (Gu et al, 2009). Briefly, for each imaging time point before (t 5 0) and after drug injection (time points indicated in the text for each experiment), mice were given 0.2 ml of luciferin at 15 mg/ml by i.p.…”

“…Mouse mdr1a is the nearest homolog to human MDR1 (Devault and Gros, 1990;Hsu et al, 1990;Tang-Wai et al, 1995), expressed predominantly in the intestine and at lower levels in liver, kidney, and spleen (Croop et al, 1989). Our reported bioimaging model has a firefly luciferase (fLUC) cDNA inserted into the murine mdr1a genetic locus by homologous recombination and we have demonstrated that abdominal luminescence intensity is an accurate reporter of intestinal mdr1a and fLUC mRNA expression in mdr1a.fLUC mice (Gu et al, 2009). The purpose of the current study was to examine the dynamic effect of repeated drug dosing on mdr1a expression and to determine the role of PXR in drug-mediated mdr1a induction in vivo.…”

“…To facilitate the study of MDR1 gene regulation in vivo, we previously reported a mouse model that allows noninvasive bioimaging of mdr1a gene expression in live animals and in real time (Gu et al, 2009). Mouse mdr1a is the nearest homolog to human MDR1 (Devault and Gros, 1990;Hsu et al, 1990;Tang-Wai et al, 1995), expressed predominantly in the intestine and at lower levels in liver, kidney, and spleen (Croop et al, 1989).…”