Imaging Cyclooxygenase-2 (Cox-2) Gene Expression in Living Animals with a Luciferase Knock-in Reporter Gene

Ishikawa, Tomo-o; Jain, Neil; Taketo, Makoto Mark; Herschman, Harvey R.

doi:10.1007/s11307-006-0034-7

Cited by 47 publications

(62 citation statements)

References 70 publications

(67 reference statements)

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“…Other luciferase knock-in reporter systems have been described (30)(31)(32), but they either lack the ability to control knock-in of the transgene via Cre/loxP recombination (30, 31) or they are not used for monitoring transcription from a regulated gene (32). With our vector design, which requires Cre-mediated recombination to achieve the fLUC knockin, we can potentially control knock-in spatially by mating mdr1a flox mice with Cre-donor mice that express Cre in a tissue-restricted way, thus limiting the mdr1a.fLUC configuration to selected tissues and organs in any given animal.…”

Section: Discussionmentioning

confidence: 99%

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

Tsark²,

Brown³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

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

Tsark²,

Brown³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Genomic fragments of the Cox-2 3 0 distal region were isolated by screening an RPCI-22 mouse bacterial artificial chromosome (BAC) library (Ishikawa et al, 2006). To construct the pCox-2-floxneo targeting vector (Fig.…”

Section: Methodsmentioning

confidence: 99%

Conditional knockout mouse for tissue-specific disruption of the cyclooxygenase-2 (Cox-2) gene

Ishikawa

Herschman

2006

genesis

Self Cite

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Cyclooxygenase-2 (Cox-2) modulates many normal functions, and appears to play a role in a wide variety of pathophysiologic conditions. Cox-2 gene expression is induced in many different cell types, in response to many distinct stimuli. We generated a conditional knockout mouse in which critical exons of the Cox-2 gene are flanked with loxP sites. Cox-2(flox/flox) mice appear normal and are fertile. Recombination at the loxP sites, loss of Cox-2 protein expression, and prevention of induced PGE2 accumulation are observed in Cox-2(flox/flox) mouse embryo fibroblasts following infection with an adenovirus expressing CRE recombinase. In vivo recombination at the Cox-2(flox) allele was demonstrated in the liver of Cox-2(flox/flox) mice following intravenous injection of adenovirus expressing CRE recombinase. Spatially and temporally restricted elimination of the Cox-2 gene in Cox-2(flox/flox) conditional knockout mice should provide a valuable tool to analyze the cell type-specific role of Cox-2 in many disease models.

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“…For example, the liver expression of luciferase in Figure 4A resulted because the CMV promoter element is "on" in hepatocytes. An alternate promoter such as cyclooxygenase 2L (cox-2L) is "off" under normal conditions in the liver, but is sensitive to being turned "on" in the presence of inflammation (Ishikawa et al 2006). The images in Figure 7 show this approach of turning luciferase from "off" to "on."…”

Section: Specialized Research Applicationsmentioning

confidence: 99%

Noninvasive Bioluminescence Imaging in Small Animals

Zinn¹,

Chaudhuri²,

Szafran³

et al. 2008

ILAR Journal

131

106

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There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

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Imaging Cyclooxygenase-2 (Cox-2) Gene Expression in Living Animals with a Luciferase Knock-in Reporter Gene

Cited by 47 publications

References 70 publications

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

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

Conditional knockout mouse for tissue-specific disruption of the cyclooxygenase-2 (Cox-2) gene

Noninvasive Bioluminescence Imaging in Small Animals

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