High-Throughput 3-D Cellular Assays Using Destabilized Green Fluorescence Protein
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Date
2008-06
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The Ohio State University
Abstract
Cell assays for high-throughput screening (HTS) of potential drug candidates are important tools in the process of drug discovery. Most cellular assays are currently based on 2-D monolayer cell cultures, but 3-D cell cultures could better mimic the in vivo characteristics of actual organism tissues. Unfortunately, assays using 3-D culture models usually require significant manual manipulation and are therefore not suitable for HTS. Research under Dr. Shang-Tian Yang has resulted in a functioning system for high-throughput 3-D cellular assays using engineered cells to express enhanced green fluorescence protein (EGFP) quantifiable through fluorometry. System improvement to allow rapid assessment of cellular events, such as specific gene expression or cell cycle progress may be limited by the relatively long persistence of the currently used reporter protein in the cells.
In this study a new fluorescence reporting cell line was established using a destabilized EGFP (d4EGFP) expressed in Chinese hamster ovary (CHO) cells. Correlating the fluorescence response with cell number for the d4EGFP expressing cell line in 2-D assays indicated that the fluorescence expression of d4EGFP may be too low for use in reporting cell number in a high-throughput manner. The fluorescence and cell number correlation in 3-D assays indicated that slightly better performance could be achieved with the d4EGFP reporter in 3-D but further testing is needed to demonstrate whether this improvement would be sufficient. Future work investigating growth and environmental conditions or further genetic modification of the cell line is recommended to possibly improve the fluorescence expression.
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Keywords
cellular assays, d4EGFP, high-throughput screening, CHO