Abstract
Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.
Highlights
Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses
In addition to its use in plants, researchers have shown biolistic delivery to be a viable strategy for delivering DNA and chemical payloads into animal cells, often penetrating skin barriers[9,10,11]
To reduce the variations caused by the different tissues and improve the measurement consistency, we employed a 3D printed double-barrel (DB) device that allows two sets of test reagents to be simultaneously bombarded in parallel into the same plant tissue
Summary
Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. In addition to its use in plants, researchers have shown biolistic delivery to be a viable strategy for delivering DNA and chemical payloads into animal cells, often penetrating skin barriers[9,10,11] Both commercial and homemade gene gun devices have been utilized to bombard DNA into plant tissues[12,13,14]. The DB device allows introduction of an internal control for each bombardment, which can be used to normalize shot-to-shot variation Another impediment to efficient optimization and quantification of biolistic delivery is counting cells transfected with the marker gene, such as the green fluorescent protein (GFP) gene used in this work. We developed a system using the double-barrel device for particle bombardment and customized CellProfiler software for cell counting This system is shown to be used effectively for the optimization of DNA transfection conditions as well as the evaluation of efficacies of CRISPR-Cas reagents
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