tetracycline and 5-bromo-4-chloro-3-indolyl-beta-galactoside

The entorhinal cortex (EC) plays a central role in episodic memory and is among the earliest sites of neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease. Given its importance in memory and dementia, the ability to selectively modulate gene expression or neuronal function in the EC is of widespread interest. To this end, several recent studies have taken advantage of a transgenic line in which the tetracycline transactivator (tTA) was placed under control of the neuropsin (Nop) promoter to limit transgene expression within the medial EC and pre-/parasubiculum. Although the utility of this driver is contingent on its spatial specificity, no detailed neuroanatomical analysis of its expression has yet been conducted. We therefore undertook a systematic analysis of Nop-tTA expression using a lacZ reporter and have made the complete set of histological sections available through the Rodent Brain Workbench tTA atlas, www.rbwb.org . Our findings confirm that the highest density of tTA expression is found in the EC and pre-/parasubiculum, but also reveal considerable expression in several other cortical areas. Promiscuous transgene expression may account for the appearance of pathological protein aggregates outside of the EC in mouse models of Alzheimer's disease using this driver, as we find considerable overlap between sites of delayed amyloid deposition and regions with sparse β-galactosidase reporter labeling. While different tet-responsive lines can display individual expression characteristics, our results suggest caution when designing experiments that depend on precise localization of gene products controlled by the Nop-tTA or other spatially restrictive transgenic drivers.

Topics: Amyloid beta-Protein Precursor; Animals; Brain; Entorhinal Cortex; Galactosides; Genes, Reporter; Humans; Immunohistochemistry; Indoles; Kallikreins; Lac Operon; Mice; Mice, Inbred C57BL; Mice, Transgenic; Promoter Regions, Genetic; Tetracycline; Trans-Activators

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2(S)) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P tight Tet-responsive promoter (P(tight)-lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P tight-lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer's disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P tight-lacZ reporter mice may be widely applicable.

Topics: Animals; Brain; Cloning, Molecular; Doxycycline; Fluorescent Antibody Technique; Galactosides; Gene Expression Regulation; Indoles; Mice; Mice, Transgenic; Neurons; Tetracycline; Thy-1 Antigens; Trans-Activators; Transgenes

A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring beta-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, beta-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteriolysis; beta-Galactosidase; Chromogenic Compounds; Enterobacteriaceae; Erythromycin; Galactosides; Hydrolysis; Indoles; Isopropyl Thiogalactoside; Microbial Sensitivity Tests; Penicillins; Peptides; Reproducibility of Results; Tetracycline