Cellular arrays for large-scale analysis of transcription factor activity
| Autor: | Beatriz Peňalver-Bernabé, Linda J. Broadbelt, Abigail D. Bellis, Angela K. Pannier, Lonnie D. Shea, Jacqueline S. Jeruss, Michael E Yarrington, Michael S. Weiss, Maria V. Barbolina |
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| Rok vydání: | 2010 |
| Předmět: |
Regulation of gene expression
Microarray analysis techniques Gene Expression Profiling Bioengineering Gene delivery Biology Microarray Analysis Applied Microbiology and Biotechnology Molecular biology Article Cell biology Gene expression profiling Gene Expression Regulation Genes Reporter Luminescent Measurements Bioluminescence imaging Luciferase Signal transduction Luciferases Transcription factor Metabolic Networks and Pathways Transcription Factors Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 108:395-403 |
| ISSN: | 0006-3592 |
| DOI: | 10.1002/bit.22916 |
| Popis: | Identifying molecular mechanisms or therapeutic targets is typically based on large-scale cellular analysis that measures the abundance of mRNA or protein; however, abundance does not necessarily correlate with activity. We report a method for direct large-scale quantification of active pathways that employs a cellular array with parallel gene delivery of constructs that report pathway activity. The reporter constructs encode luciferase, whose expression is influenced by binding of transcription factors (TFs), which are the downstream targets of signaling pathways. Luciferase levels are quantified by bioluminescence imaging (BLI), which allows for rapid, non-invasive measurements. Activity profiles by BLI of 32 TFs were robust, consistent, and reproducible, and correlated with standard cell lysis techniques. The array identified five TFs with differential activity during phorbol-12-myristate-13-acetate (PMA)-induced differentiation of breast cancer cells. A system for rapid, large-scale, BLI quantification of pathway activity provides an enabling technology for mechanistic studies of cellular responses and processes. |
| Databáze: | OpenAIRE |
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