The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regula- A "genetic switch" that could be operated at will and that would permit the control of individual gene activities quantitatively and reversibly in a temporal and spatial manner would thus be of great advantage. The tetracycline (Tc)-controlled systems for the activation of transcription (7, 8) fulfill a number of these requirements at the cellular level. Herein, we report that the "reverse Tc-controlled transactivator" (rtTA) system, where doxycycline (dox) acts as an inducer of transcription as well as the "Tc-controlled transactivator" (tTA) system, where Tc or dox prevent transcription activation (Fig. 1) can be operated in a quantitative and highly tissue-specific way when transferred into mice. The results show that the controls are tight and that the kinetics of induction, especially with the rtTA system, are rapid. Although we (9, 10) and others (11,12) have reported that the tTA system can be applied to transgenic organisms, the results reported herein establish that both the rtTA and the tTA systems provide true genetic switches capable of quantitatively controlling individual gene activities in animals in a highly tissue-specific manner. These observations open up exciting prospects for the study of gene function in mammalian organisms. Transgenic Animals. Transgenic mouse lines (NMRI outbred) were generated by pronuclear injection using standard techniques (14) and analyzed by the Southern blot technique (15) using the BamHI-EcoRV fragment of the luciferase gene and the XbaI fragment of the tTA gene as respective probes. Luciferase reporter mice were obtained upon transfer of the 3.1-kb XhoI-EaeI fragment of pUHC13-3 (7). Mice producing rtTA controlled by PhCMV, the promoter of the human cytomegalovirus immediate early genes, were obtained upon transfer of the 2.7-kb PflMI-XhoI fragment and animals synthesizing tTA under the control of the LAP promoter were obtained by transferring the 5.5-kbAseI-Asp700 fragment of pUHG15-30. Animals transgenic for both a transactivator and a reporter unit were exposed when necessary to doxycycline hydrochloride (Sigma) dissolved in 5% sucrose supplied as drinking water, which was exchanged every 3 days. Possible long-term effects of dox (200 ,ug/ml) were exam...

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

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Galantamine (Reminyl), an approved treatment for Alzheimer's disease (AD), is a potent allosteric potentiating ligand (APL) of human ␣3␤4, ␣4␤2, and ␣6␤4 nicotinic receptors (nAChRs), and of the chicken/mouse chimeric ␣7/5-hydroxytryptamine 3 receptor, as was shown by whole-cell patch-clamp studies of human embryonic kidney-293 cells stably expressing a single nAChR subtype. Galantamine potentiates agonist responses of the four nAChR subtypes studied in the same window of concentrations (i.e., 0.1-1 M), which correlates with the cerebrospinal fluid concentration of the drug at the recommended daily dosage of 16 to 24 mg. At concentrations Ͼ10 M, galantamine acts as an nAChR inhibitor. The other presently approved AD drugs, donepezil and rivastigmine, are devoid of the nicotinic APL action; at micromolar concentrations they also block nAChR activity. Using five CHO-SRE-Luci cell lines, each of them expressing a different human muscarinic receptor, and a reporter gene assay, we show that galantamine does not alter the activity of M1-M5 receptors, thereby confirming that galantamine modulates selectively the activity of nAChRs. These studies support our previous proposal that the therapeutic action of galantamine is mainly produced by its sensitizing action on nAChRs rather than by general cholinergic enhancement due to cholinesterase inhibition. Galantamine's APL action directly addresses the nicotinic deficit in AD.

TGR5 (Gpbar-1) is a plasma membrane-bound, G protein-coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11-positive recycling endosome of gallbladder epithelial cells.