期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 98, 期 2, 页码 486-497出版社
WILEY
DOI: 10.1002/bit.21477
关键词
transfected cell array; bioluminescence imaging; substrate-mediated gene delivery; estrogen receptor; breast cancer
资金
- NCI NIH HHS [R21 CA125285-01, P50 CA089018, P50 CA 89018, R21 CA125285] Funding Source: Medline
- NIGMS NIH HHS [R01 GM066830-01A1, R01 GM066830] Funding Source: Medline
Transfected 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 (ER alpha) activity in MCF-7 breast cancer cells. A plasmid containing an ER alpha-regulated promoter directing firefly luciferase expression was mixed with a normalization plasmid, complexed with cationic lipids and deposited into an array. 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.
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