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High-Throughput Methods for Screening Polymeric Transfection Reagents

2013; Cold Spring Harbor Laboratory Press; Volume: 2013; Issue: 11 Linguagem: Inglês

10.1101/pdb.prot078634

1940-3402

Gregory T. Zugates, Daniel G. Anderson, Róbert Langer,

Virus-based gene therapy research

The clinical success of gene therapy requires the development of a safe and efficient delivery system for DNA. Cationic polymers are nonviral vectors that can associate electrostatically with plasmid DNA to form nanocomplexes. In some cases, this is sufficient for cellular uptake and transfection, although the precise mechanisms by which polymers facilitate gene delivery remain unclear. A robust and reliable method to screen for efficacy is essential for the development of effective polycationic transfection reagents. Numerous parameters must be controlled and optimized, such as polymer structure, polymer-DNA-binding conditions (mixing method, pH, ionic strength, incubation time, concentrations, ratios), transfection media (type, serum content), DNA dose and incubation time with the cells, cell specificity, and assay conditions. In this protocol, we describe a high-throughput method for assessing polymer-mediated transfection. The technique uses 96-well plates, which allows many transfection parameters to be varied and optimized in parallel. Hundreds of polymers can be tested in quadruplicate in a single day and the technique can easily be automated to efficiently and reproducibly test large material libraries. One limitation is that many plate types, solutions, and equipment must be stocked and sterilized. Moreover, because all polymers are processed simultaneously in very small volumes, it is difficult to validate each step for each polymer to ensure solution uniformity and adequate polymer-DNA complexation. Despite these drawbacks, this high-throughput screening method has already been used successfully in the development of efficient polycation vectors.