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Transfection

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Transfection is the process of introducing nucleic acids into cells by non-viral methods.[1] The term "transformation" is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells; "transduction" is often used to describe virus-mediated DNA transfer.

Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Transfection can result in unexpected morphologies and abnormalities in target cells.

Terminology

The meaning of the term has evolved.[2] The original meaning of transfection was "infection by transformation", i.e. introduction of DNA (or RNA) from a eukaryote-infecting virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA.

Methods

There are various methods of introducing foreign DNA into a eukaryotic cell. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles.

One of the cheapest methods is transfection by calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[3] (see also [4]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell. A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e. small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, lipid-cation based transfection is more typically used, because the cells are more sensitive.

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.

A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus. DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and the cells are said to be transduced.

Magnet assisted transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid particle complexes towards and into the target cells, where the cargo is released.[5][6][7]

Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focussed laser. This technique was first described in 1984 by Tsukakoshi et al, who used a frequency tripled Nd:YAG to generate stable and transient transfection of normal rat kidney cells[8]. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.

Other methods of transfection include nucleofection, electroporation, sonoporation, heat shock, magnetofection and proprietary transfection reagents such as Lipofectamine, PromoFectin, Dojindo, GenePORTER, Hilymax, Fugene, jetPEI, Effectene or DreamFect.

Stable and transient transfection

For most applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded.

If it is desired that the transfected gene actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. To accomplish this, a marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able to proliferate, while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further.

A common agent for stable transfection is Geneticin, also known as G418, which is a toxin that can be neutralized by the product of the neomycin resistant gene.

See also

References

  1. ^ http://www.promega.com/paguide/chap12.htm
  2. ^ "Transfection" at Dorland's Medical Dictionary
  3. ^ Graham FL, van der Eb AJ (1973). "A new technique for the assay of infectivity of human adenovirus 5 DNA". Virology. 52 (2): 456–67. doi:10.1016/0042-6822(73)90341-3. PMID 4705382.
  4. ^ Bacchetti S, Graham F (1977). "Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA". Proc Natl Acad Sci USA. 74 (4): 1590–4. doi:10.1073/pnas.74.4.1590. PMID 193108.
  5. ^ Bertram, J. (2006) MATra - Magnet Assisted Transfection: Combining Nanotechnology and Magnetic Forces to Improve Intracellular Delivery of Nucleic Acids. Current Pharmaceutical Biotechnology 7, 277-28
  6. ^ http://www.magnet-assisted-transfection.com/naps/naps_fr02_01.html
  7. ^ http://www.promokine.info/products/cell-transfection/magnet-assisted-transfection/
  8. ^ M. Tsukakoshi, S. Kurata, Y. Nomiya, et al., "A Novel Method of DNA Transfection by Laser Microbeam Cell Surgery". Applied Physics B-Photophysics and Laser Chemistry. 35(3): 135-140 (1984)
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