Bioluminescence imaging for assessment and normalization in transfected cell arrays
Biotechnol Bioeng
A. K. Pannier, E. A. Ariazi, A. D. Bellis, Z. Bengali, V. C. Jordan, L. D. SheaTransfected cell arrays (TCAs) represent a high-throughput technique to correlate gene expression with functional cell responses. Despite advances in TCAs, improvements are needed for the widespread application of this technology. We have developed a TCA that combines a two-plasmid system and dual-bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The two-plasmids consist of: i) normalization plasmid present within each spot, and ii) functional plasmid that varies between spots, responsible for the functional endpoint of the array. Bioluminescence imaging of dual-luciferase reporters (renilla, firefly luciferase) provides sensitive and quantitative detection of cellular response, with minimal post-transfection processing. The array was applied to quantify estrogen receptor alpha (ERalpha) activity in MCF-7 breast cancer cells. A plasmid containing an ERalpha-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and spotted into an array using soft lithography techniques. ER induction mimicked results obtained through traditional assays methods, with estrogen inducing luciferase expression 10-fold over the antiestrogen fulvestrant or vehicle. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. This system provides a tool for basic science research, with potential application for the development of patient specific therapies. (c) 2007 Wiley Periodicals, Inc.
2007
Biotechnol Bioeng
