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...

Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.

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The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.

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.