Cellular Arrays (US Patent Application)

The present invention relates to characterizing transcription within cells. In particular, the present invention provides transfected cell arrays (e.g., two-dimensional and/or three-dimensional arrays) and systems, kits and methods utilizing the same (e.g., for transcriptional activity characterization). Compositions and methods of the present invention find use in, among other things, research, drug discovery and clinical (e.g., diagnostic, preventative and therapeutic) applications

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 α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-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

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 α (ERα) activity in MCF-7 breast cancer cells. A plasmid containing an ERα-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

Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis.Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation.G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen

Cellular Arrays (US Patent Application)

The present invention relates to characterizing transcription within cells. In particular, the present invention provides transfected cell arrays (e.g., two-dimensional and/or three-dimensional arrays) and systems, kits and methods utilizing the same (e.g., for transcriptional activity characterization). Compositions and methods of the present invention find use in, among other things, research, drug discovery and clinical (e.g., diagnostic, preventative and therapeutic) applications

Substrate-Mediated Gene Delivery for Assessment of Signal Transduction Pathways in Cancer Cells

Gene delivery has the potential to be used in diagnostic applications, specifically to investigate cellular signal transduction pathways responsible for disease. Analysis of multiple pathways or genes in a parallel format can be achieved using a transfected cell array, a high throughput approach to correlate gene expression with functional cell responses, based on gene delivery from a substrate that supports cell adhesion. Substrate-mediated gene delivery functions by self-assembling DNA with nonviral vectors, resulting in positively charged complexes that can interact with a biomaterial or substrate. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. DNA complexes can be immobilized on the substrate through specific interactions introduced through complementary functional groups on the vector and surface or through nonspecific interactions. As surface properties are critical to the efficiency of the surface delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to study the mechanisms of transfection by complexes nonspecifically immobilized on chemically specific substrates. Surface hydrophilicity and ionization were found to mediate both immobilization and transfection. Additionally, SAMs were used in conjugation with soft lithographic techniques to imprint substrates with specific patterns of SAMs, resulting in patterned DNA complex deposition and transfection

Transfected Cell Arrays for Assessment of Estrogen Receptor Activation in Breast Cancer Cells

Transfected cell arrays represent a high-throughput approach to correlate gene expression with functional cell responses, based on gene delivery from a substrate that supports cell adhesion. These arrays provide the ability to express, in parallel, thousands of exogenous genes in live cells, giving real-time information on cellular physiology and gene function. While there have been advances in transfected cell arrays, improvements are needed to this technology, including increasing the cells types that can be efficiently transfected and developing better quantification and normalization methods. We have created an array using soft lithography techniques to pattern DNA-lipid complex deposition. Specifically, a mold was fabricated by curing polydimethylsiloxane (PDMS) into thin, flat disks. Rods of precise diameters were then used to punch holes into the mold, with diameters ranging from 1 mm to 3 mm. The PDMS mold was oxidized and then reversibly sealed to polystyrene slides. The holes in the mold, termed microwells, served as reservoirs for complex deposition onto the polystyrene slides. After deposition, the PDMS mold was peeled away from the polystyrene slide, which was then rinsed thoroughly. DNA complexes were immobilized on the slide in distinct regions, replicating the pattern of microwells in the PDMS mold. Transfection of cells seeded onto these slides was also confined to the patterns