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Generation of magnetic nonviral gene transfer agents and magnetofection in vitro

Nature Protocols volume 2, pages 2391–2411 (2007)Cite this article

Abstract

This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force–assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3–5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.

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Figure 1
Figure 2: Radiolabeling (iodination) of plasmid DNA and testing DNA association with magnetic nanoparticles in transfection complexes.
Figure 3: Enhanced GFP (eGFP) reporter gene expression and cell association of transfection complexes comprising magnetic nanoparticles with H441 human lung epithelial cells detected by microscopy.
Figure 4: Magnetofection versus lipofection efficiency.
Figure 5: Magnetofection versus lipofection efficiency in H441 human lung epithelial cells.
Figure 6: Magnetofection versus lipofection efficiency in Jurkat cells.

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Acknowledgements

The authors would like to thank E. Hammerschmid for the help with the flow cytometry analysis, and Dr. Joachim Aigner for the help with microscopy studies and valuable discussions. 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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  1. Institute of Experimental Oncology, Technische Universität München, Ismaninger Strasse 22, Munich, 81675, Germany

    Olga Mykhaylyk, Yolanda Sánchez Antequera, Dialekti Vlaskou & Christian Plank

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Correspondence to Olga Mykhaylyk.

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Christian Plank is a co-founder of OZ Biosciences, Marseille, France. OZ Biosciences manufactures and sells magnetofection reagents.

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Mykhaylyk, O., Antequera, Y., Vlaskou, D. et al. Generation of magnetic nonviral gene transfer agents and magnetofection in vitro. Nat Protoc 2, 2391–2411 (2007). https://doi.org/10.1038/nprot.2007.352

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