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A new model for studying tissue-specific mdr1a 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.

SUBMITTER: Gu L

PROVIDER: S-EPMC2654810 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them.ResultsTo facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration.ConclusionsThe ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.

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A new model for studying tissue-specific mdr1a gene expression in vivo by live imaging.

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