UB - University at Buffalo, The State University of New York

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Quantification of gene expression dynamics is limited due to the destructive, expensive and laborious nature of current gene expression profiling techniques such as qRT-PCR and cDNA microarrays. Here we developed scalable live-cell microarrays to measure gene expression dynamics in real time and in a high-throughput manner. To this end, we generated dual-promoter lentiviral vectors that were designed to provide independent and high level gene expression. Each lentivirus harbored a transcriptional regulatory element e.g. NF-ï?«B or promoter e.g. IL-8p encoding for destabilized green fluorescence protein and a constitutive promoter driving red fluorescence protein for signal normalization. Lentivirus preparations were immobilized in a microarray format using a robotic spotter to generate the LentiVirus microArray (LVA). Target cells were transduced with immobilized lentivirus and after treatment with TNF-ï?¡, IL-1 or IFN-ï?§ transcriptional activity was interrogated in real-time using fluorescence microscopy. In contrast to standard methods, our experiments provided rich dynamic information over a period of several days. Data normalization by red fluorescence intensity eliminated errors due to spot-to-spot variability in transduction efficiency or changes in cell proliferation upon cytokine treatment. These results were confirmed by flow cytometry. Finally, contrary to transfection arrays, the LVA can monitor gene expression in primary cells and stem cells thereby providing a useful tool for deciphering gene regulatory networks of complex biological processes.