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Parallel Gene Delivery for Cell-Based Activity Assays

Abigail D. Bellis and Lonnie D. Shea. Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E136, Evanston, IL 60208

Many molecular markers have been inadequate for identifying therapeutic targets or predicting patient outcomes, which has motivated the development of cell-based assays that provide the physiological context in which to investigate molecular function. Gene delivery, in particular, is being employed with these assays to manipulate a specific factor within the cell. In previous proof of principle work, we have shown the ability to quantify cellular activity on a small scale within the format of a transfected cell array. Current efforts are focused on developing the methods to scale up this system to facilitate broader functional analysis. We are investigating the use of parallel gene delivery for high throughput cell based assays using either a slide or well-plate format. Achieving adequate transfection while maintaining high cell viability poses a significant challenge to automating this approach. Transfection of the luciferase encoding plasmid was analyzed using bioluminescent imaging. Bioluminescent imaging provides the ability to sensitively quantify transgene expression while limiting post-transfectional processing. For slides, plasmids are spotted at room temperature at a relative humidity of 85% in an array format onto the slide surface. The small volume of DNA/lipid complexes used during spotting complicates transfection, as uncontrolled surface adsorption or dehydration can diminish lipoplex activity. The addition of sucrose can protect and stabilize DNA/lipid complexes. Transfection in the presence of sucrose was increased four-fold relative to its absence. Slide incubation time before cell seeding was also investigated. Longer incubation times resulted in more complexes immobilized onto the spot surface. The correlation between transfection efficiency and incubation time was found to be dependent on initial DNA concentration within the spot. Multi-well plates were also investigated as an alternative to slides. Transfection within the plates was more consistent than on the slides yet requires more advanced automation for cell seeding and data acquisition. Because of the slightly lower transfection variability the plate format offered an important tool to determine proper experimental conditions for implementation within the transfected cell array. The ability to transfect cells with high efficiency in an array format is a fundamental tool that can be used in numerous cell-based screening assays.