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M. C. Butler, J. M. Sullivan; A Versatile Instrument for High Throughput and High Content Screening in Gene-Based Drug Discovery. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5342.
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© ARVO (1962-2015); The Authors (2016-present)
Develop a quantitative imaging platform (QIP) for high throughput (HTP) and high content screening (HCS) of transfected cells to identify highly active post-transcriptional gene silencing (PTGS) agents such as ribozymes.
A Nikon TE300 microscope was extensively modified with a variety of optical excitation paradigms, and for a variety of fluors to acquire steady state and real time optical data from fluors used to quantitate expression levels of target proteins and RNAs in cells transfected in 96-well dishes. Optical cubes were designed for SYTOX Blue, Turbo yellow fluorescent protein (TYFP), Alexa-647, and Alexa-750. These fluors are used for measure of cell number, transfection efficiency, target protein, and target RNA, respectively, in cells. The Alexa fluors were selected to eliminate any contribution of cellular auto fluorescence to the measures, and SYTOX Blue and TYFP were selected for ratiometric normalization of target protein or mRNA to cell number and transfection efficiency. Generally, 4X images were used to acquire quantitative data from an ensemble of transfected cells. However, single cell data was acquired using a 20X lens. The entire data acquisition environment including the robotic stage use closed loop computer controls (IP Labs) and the z-axis was programmed for auto-focus using in-house scripts.
HEK293 cells were co-transfected with full length target, and hammerhead ribozyme expression constructs in 96-well dishes. Cell counting by SYTOX Blue was linear over several log orders, and TYFP measured transfection efficiency on a well-by-well basis. Monoclonal antibody against the target was labeled with NHS-Alexa-647 and used in a primary antibody screen assay to quantify the target protein directly. A second monoclonal against another target labeled with NHS-Alexa-750 allows for multi-target protein measures, or the Alexa-750 channel could be used to measure target RNA. The RNase-resistant antisense probes were made with T7 polymerase and amino-allyl-UTP and coupled to NHS-Alexa-750. Knockdown of target mRNAs and proteins was measured quantitatively and normalized to either or both cell number and transfection efficiency on a well-by-well basis. Protein and RNA quantitation by the QIP were compared to standard protein or RNA blotting experiments.
This is a robust, novel, and useful QIP for HTP and HCS of transfected cells for PTGS drug discovery. The current device has the capacity for HTS and HCS in a 96-well dish. Plate robotics will realize the potential of the QIP for combinatorial library screening.
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