Bioluminescence imaging for assessment and normalization in transfected cell arrays.
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 (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 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.
Pannier
Angela K
AK
Department of Interdepartmental Biological Sciences, Northwestern University, Evanston, Illinois.
Ariazi
Eric A
EA
Bellis
Abigail D
AD
Bengali
Zain
Z
Jordan
V Craig
VC
Shea
Lonnie D
LD
D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D006801:Humans; D049409:Luciferases, Firefly; D049410:Luciferases, Renilla; D008163:Luminescent Measurements / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010957:Plasmids; D012680:Sensitivity and Specificity; D049490:Systems Biology; D014162:Transfection / Q000379:methods*
D047628:Estrogen Receptor alpha; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly