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...

Alzheimer's disease (AD) is characterized by presence of extracellular fibrillar Aβ in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble Aβ oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing Aβ oligomers from AD brains, but not by an extract from non-AD brains. Aβ oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Levels of amyloid-beta monomer and deposited amyloid-beta in the Alzheimer’s disease brain are orders of magnitude greater than soluble amyloid-beta oligomer levels. Monomeric amyloid-beta has no known direct toxicity. Insoluble fibrillar amyloid-beta has been proposed to be an in vivo mechanism for removal of soluble amyloid-beta and exhibits relatively low toxicity. In contrast, soluble amyloid-beta oligomers are widely reported to be the most toxic amyloid-beta form, both causing acute synaptotoxicity and inducing neurodegenerative processes. None of the amyloid-beta immunotherapies currently in clinical development selectively target soluble amyloid-beta oligomers, and their lack of efficacy is not unexpected considering their selectivity for monomeric or fibrillar amyloid-beta (or both) rather than soluble amyloid-beta oligomers. Because they exhibit acute, memory-compromising synaptic toxicity and induce chronic neurodegenerative toxicity and because they exist at very low in vivo levels in the Alzheimer’s disease brain, soluble amyloid-beta oligomers constitute an optimal immunotherapeutic target that should be pursued more aggressively.