Nucleofection

From Wikipedia, the free encyclopedia

Nucleofection is a transfection method which enables efficient and reproducible transfer of nucleic acids such as DNA, RNA, Small interfering RNA into cells so far considered difficult or even impossible to transfect. Nucleofection, also referred to as Nucleofector Technology was invented by amaxa (http://www.amaxa.com). “Nucleofector” and “nucleofection” are trademarks, owned by amaxa AG.


[edit] Applications

Nucleofection is an efficient method to transfer substrates into mammalian cells. Examples for such substrates are nucleic acids, like the DNA of an isolated gene Molecular cloning into a plasmid or Small interfering RNA for knocking down expression of a specific endogenous gene.

Especially primary cells, for example stem cells, fall into this category, but also many cell lines are difficult to transfect. Primary cells are freshly isolated from body tissue and thus cells are unchanged, closely resembling the in-vivo situation, and are therefore of particular relevance for medical research purposes. In contrast, cell lines have often been cultured for decades and may significantly differ from their origin.

[edit] Mechanism

Based on the physical method of electroporation, nucleofection uses a combination of optimized electrical parameters, generated by a special device called Nucleofector, with cell-type specific reagents. The substrate is transferred directly into the cell nucleus and the cytoplasm. In contrast, other commonly used non-viral transfection methods rely on cell division for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even non-dividing cells, such as neuron and resting blood cell.Before the introduction of the Nucleofector Technology, efficient gene transfer into primary cells had been restricted to the use of viral vectors, which typically involve disadvantages, such as, safety risks, lack of reliability, and high cost. The non-viral gene transfer methods available were not suitable for the efficient transfection of primary cells. All of the non-viral methods around required cell division for completion of transfection, since the DNA only enters the nucleus to a sufficient level during breakdown of the nuclear envelope upon cell division. The Nucleofector Technology, however has changed all this and has made these primary cells, with limited or no ability to divide, accessible for efficient gene transfer. This enables researchers to study gene functions in a situation comparable to body tissue and it provides far-reaching approaches for scientific research and therapeutic development. Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, RNA, siRNAs, shRNAs, mRNAs and pre-mRNAs, BACs, peptides, morpholinos, PNA, or other biologically active molecules. Hence switching between substrates or performing co-transfections is straightforward.

[edit] References

  • Efficient gene transfer into the human natural killer cell line, NKL, using the amaxa

nucleofection system Journal of Immunological Methods; 2004 Jan; 284(1-2):133-40 Kerima Maasho, Alina Marusina, Nicole M. Reynolds, John E. Coligan, and Francisco Borrego PMID: 14736423

  • Effective gene delivery to adult neurons by a modified form of electroporation

J Neurosci Methods; 2005 Mar 15; 142(1):137-43 Pascal G. Leclere, Aliza Panjwani, Reginald Docherty, Martin Berry, John Pizzey, and David A. Tonge PMID: 15652627

  • Nucleofection is an efficient non-viral transfection technique for human bone marrowderived mesenchymal stem cells Stem Cells; Vol. 24 No. 2; February 2006, pp. 454-461 Michela Aluigi, Miriam Fogli, Antonio Curti, Alessandro Isidori, Elisa Gruppioni, Claudia Chiodoni, Mario P. Colombo, Piera Versura, Antonia D’Errico-Grigioni, Elisa Ferri, Michele Baccarani, and Roberto M. Lemoli

PMID: 16099993

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