Bioluminescence imaging for assessment and normalization in transfected cell arrays

Abstract

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  (ER) activity in MCF-7 breast cancer cells. A plasmid containing an ER-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.

Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

“…Atomic force microscopy imaging of immobilized complexes supports this hypothesis, revealing that the presence of PEG on the surface significantly affects the morphology of the complexes, which correlates with greater high transfection levels and transfection efficiencies. The ability to control the morphology of the immobilized complexes and thus influence transfection levels could be translated to scaffolds for gene delivery in tissue engineering applications [5,6], as well other applications of substrate-mediated gene delivery, including transfected cell arrays [63]. Cell adhesion on EG-containing SAMs.…”

Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

“…There have been many efforts to increase the efficiency of nonviral complex delivery, including alternative delivery strategies like substrate-mediated gene delivery (SMD). SMD is a technique where DNA-NPs are immobilized to substrates, and cells are then seeded onto these DNA-NPs [7][8][9][10]. SMD is often compared and contrasted to bolus gene delivery, amore traditional gene delivery technique where DNA-NP complexes are simply pipetted into cell culture media and allowed to diffuse to the seeded cell layer.…”

Identifying Intracellular pDNA Losses From a Model of Nonviral Gene Delivery

“…The desired outcome when cells are exposed to the pDNA packets is for each cell to direct the pDNAs to the cell nucleus and produce the protein in relevant levels for applications in gene therapeutics [61], [62], diagnostics and functional genomics [63], [64], medical devices [65], and tissue engineering [66], [67]. However, the primary transmission as described (Fig.…”