Abstract
This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells in vitro by magnetic force–assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection, and association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.
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Acknowledgments
The authors would like to thank Dr. Bob Scholte for transduction of the H441 cells with eGFP and luciferase using lentiviral vectors. This work was supported by the European Union through the FP6-LIFESCIHEALTH Project “Improved precision of nucleic acid based therapy of cystic fibrosis” under contract no. 005213 as well as by the German Ministry of Education and Research, Nanobiotechnology grants 13N8186 and 13N8538. Financial support of the German Excellence Initiative via the “Nanosystems Initiative Munich (NIM)” is gratefully acknowledged.
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Mykhaylyk , O., Zelphati , O., Hammerschmid , E., Anton , M., Rosenecker , J., Plank , C. (2009). Recent Advances in Magnetofection and Its Potential to Deliver siRNAs In Vitro. In: Sioud, M. (eds) siRNA and miRNA Gene Silencing. Methods in Molecular Biology, vol 487. Humana Press. https://doi.org/10.1007/978-1-60327-547-7_6
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DOI: https://doi.org/10.1007/978-1-60327-547-7_6
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