High‐throughput ‘On Chip’ Protein and Nucleic Acid Transfection

Abstract

Transfection describes the nonviral introduction of exogenous molecules into eukaryotic cells. Numerous transfection methods have been developed to transfer nucleic acids, proteins and other macromolecules across the plasma membrane efficiently. These include physical methods, such as electroporation, magnetofection and microinjection, as well as a chemical or biological carrier‐mediated methods. Chemical transfection reagents such as cationic lipids or polymers are widely used, either alone or in combination with scaffolds. Biological methods include delivery with cell‐penetrating protein domain fusions such as trans‐activator of transcription protein from human immunodeficiency virus, VP22 or Antennapedia peptides. Certain proteins such as zinc‐finger nucleases, which are used for targeted genome modification, even appear to have intrinsic cell‐penetrating properties. To facilitate large‐scale genomic and proteomic studies there is an increasing need for automated high‐throughput transfection platforms. Several such platforms have been developed, including multiwell plates, transfection microarrays and microfluidic chip formats. Overall, the different advantages and applications of these diverse methods to deliver cargo into cells are discussed.

Key Concepts:

  • Cell membranes are a barrier to the delivery of proteins and nucleic acids into cells.

  • Mechanical, chemical and biological methods of transfection have been developed to cross the cell membrane barrier.

  • Positively charged molecules, such as cationic lipids, polymers or peptides, can help to penetrate the negatively‐charged surface of cell membranes.

  • Various automatable high‐throughput technologies have been developed, including transfection microarrays.

  • Reverse transfection is a scalable, potentially high‐throughput process where cells are added to transfectable molecules, preimmobilised on a scaffold.

Keywords: transfection; protein transfection; reverse transfection; cell microarray; gene delivery

Figure 1.

Overview of methods of delivery of nucleic acids and proteins across cell membranes.

Figure 2.

The bead transfection method (Isalan et al., ). Streptavidin‐coated Dynabeads (3 m diameter) bind to the biotinylated molecules that are to be transfected (e.g. PCR DNA, siRNA or protein). Coated beads are incubated with transfection reagent and exposed to the cells. In the example to the right, a PCR product encoding the EGFP gene under a CMV promoter is made with a biotinylated primer; a single bead is shown transfecting a single cell within a lawn of MDCK cells.

Figure 3.

The principle of reverse transfection microarrays (Ziauddin and Sabatini, ). The transfected macromolecule is spotted onto a glass slide and is subsequently coated with transfection reagent and then cells. The spots result in locally transfected fields of cells which can be analysed by microscopy.

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Valero A, Post JN, van Nieuwkasteele JW et al. (2008) Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab on a Chip 8: 62–67.

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Herrmann, Frank, and Isalan, Mark(Apr 2014) High‐throughput ‘On Chip’ Protein and Nucleic Acid Transfection. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020899.pub2]