Author:
Miller Kyle,Eggenberger Alan L.,Lee Keunsub,Liu Fei,Kang Minjeong,Drent Madison,Ruba Andrew,Kirscht Tyler,Wang Kan,Jiang Shan
Abstract
AbstractBiolistic 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.
Funder
U.S. Department of Agriculture
American Chemical Society Petroleum Research Fund
Publisher
Springer Science and Business Media LLC
Cited by
22 articles.
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