Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.
Journal - BioTechniques (United States )
beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.
|ISSN : ||0736-6205|
|Mesh Heading : ||Animals Enzyme Activation Erythrocytes Hemoglobins Lac Operon Luminescent Measurements Molecular Biology Sensitivity and Specificity beta-Galactosidase methods metabolism|
|Mesh Heading Relevant : ||Genes, Reporter metabolism metabolism genetics|
In vivo antagonism of AhR-mediated gene induction by 3'-methoxy-4'-nitroflavone in TCDD-responsive lacZ mice.
Journal - Toxicological sciences : an official journal of the Society of Toxicology (United States )
The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a member of the bHLH-PAS family of proteins. The highest-affinity ligand of this receptor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent immunological, reproductive, and developmental toxicant. The mechanism of TCDD-induced toxicity and the gene modulations that result in toxicity have not been fully defined. The majority of work to date exploring AhR function has focused on agonist-activated AhR signaling. However, it is expected that a better understanding of AhR antagonism will lead to an improved understanding of TCDD toxicity and other AhR-mediated events. This study contributes to such investigations by utilizing the AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) and a dioxin-responsive lacZ transgenic mouse model to characterize antagonism of the receptor system in vivo. The dose-response and time course of TCDD-induced transgene activation were evaluated in transgenic mice to provide information necessary to design 3'M4'NF in vivo studies. TCDD induction of the transgene was noted as early as 8 h after exposure in the lung. 3-miccrog/kg body weight TCDD was the lowest dose found to induce the reporter transgene. Finally, experiments were performed to evaluate the in vivo efficacy of 3'M4'NF. We found that 3'M4'NF inhibits TCDD-mediated reporter gene activation and CYP1A1 induction in vivo. Based on these findings, it is clear that DRE-lacZ animals and the antagonist 3'M4'NF represent important tools which will help in the identification of tissues where AhR is active, and to further characterize AhR-mediated signaling.
|ISSN : ||1096-6080|
|Mesh Heading : ||Animals Blotting, Western Dose-Response Relationship, Drug Drug Interactions Flavonoids Gene Expression Regulation Genes, Reporter Lac Operon Male Mice Mice, Inbred C57BL Mice, Transgenic Receptors, Aryl Hydrocarbon Tetrachlorodibenzodioxin Time Factors Transcriptional Activation beta-Galactosidase blood genetics drug effects|
|Mesh Heading Relevant : ||pharmacology drug effects drug effects genetics toxicity|