Transfection

Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods [1] in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells - a distinctive sense of transformation refers to spontaneous genetic modifications (mutations to cancerous cells (Carcinogenesis), or under stress (UV irradiation)). "Transduction" is often used to describe virus-mediated DNA transfer. The word transfection is a blend of trans- and infection.

Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected.

Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. 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: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers.

Chemical-based transfection

Chemical-based transfection can be divided into several kinds: cyclodextrin[3], polymers[4], liposomes, or nanoparticles [5] (with or without chemical or viral functionalization. See below).

* One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[6] (see also [7]). 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, transfection is better achieved using cationic lipids (or mixtures), because the cells are more sensitive. A popular agent was DOPA, and now more effectively Lipofectamine and UptiFectin [8].

* 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. Popular agents of this type are the Fugene [9] or LT-1 [10], and JetPEI [11].

* Other proprietary chemical transfection reagents: PromoFectin, GenePORTER, Hilymax [12]. Effectene or Altogen's cell line specific reagents.


Non chemical methods

* Electroporation is a popular method, although requiring an instrument and affecting the viability of many cell types, that also creates micro-sized holes transiently in the plasma membrane of cells under an electric discharge.

* Similarly, transfection applying sonic forces to cells, referred as Sono-poration.

* 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[13]. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.

* Gene electrotransfer is a technique that enables transfer of genetic material into prokaryotic or eukaryotic cells. It is based on a physical method named electroporation, where transient increase in the permeability of cell membrane is achieved when submitted to short and intense electric pulses.


Particle-based methods

* 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.

* Magnetofection, or 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.[14][15][16]

* Impalefection is carried out by impaling cells by elongated nanostructures such as carbon nanofibers or silicon nanowires which have been functionalized with plasmid DNA.


Viral methods

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.

Other (and hybrid) methods

Other methods of transfection include nucleofection, heat shock.

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

RNA transfection

RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. The later application is referred as siRNA transfection or RNA silencing, and has become a major application in research (to replace the "knock-down" experiments, to study the expression of proteins, i.e. of Endothelin-1 [17]) with potential applications in gene-therapy.

A limitation of the silencing approach rely on the toxicity of the transfection for cells, and its suspected effect on the expression of other genes/proteins.

See also

* Protofection
* Transformation
* Transduction
* Cationic liposome
* Nucleofection
* Magnet assisted transfection


References

1. ^ http://www.promega.com/paguide/chap12.htm
2. ^ Transfection at Dorland's Medical Dictionary
3. ^ Menuel S, Fontanay S, Clarot I, Duval R.E, Diez L, Marsura A (2008). "Synthesis and Complexation Ability of a Novel Bis- (guanidinium)-tetrakis-(β-cyclodextrin) Dendrimeric Tetrapod as a Potential Gene Delivery (DNA and siRNA) System. Study of Cellular siRNA Transfection". Bioconjugate Chem. 19 (12): 2357–2362. doi:10.1021/bc800193p. PMID 19053312.
4. ^ Fischer D, von Harpe A, Kunath K, Petersen H, Li YX, Kissel T (2002). "Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes". Bioconjugate Chem. 13 (5): 1124–1133. doi:10.1021/bc025550w.
5. ^ http://www.transfection.ws/nanoparticle_based_transfection_reagents
6. ^ 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.
7. ^ 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.
8. ^ from Interchim
9. ^ from Roche
10. ^ from Mirus bio
11. ^ from PolyPlus
12. ^ from Dojindo
13. ^ 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)
14. ^ 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
15. ^ http://www.magnet-assisted-transfection.com/naps/naps_fr02_01.html
16. ^ http://www.promokine.info/products/cell-transfection/magnet-assisted-transfection/
17. ^ Mawji et al used this technique to evaluate the kinetics of Endothelin-1 RNA decay in primary endothelial cells: Mawji et al. "RNA transfection is a versatile tool to investigate endothelin-1 posttranscriptional regulation." Exp Biol Med. 2006 Jun;231(6):704-8. PMID 16740984


External links

* MeSH Transfection
* Biology Research Resource - Articles and Forums about Transfection
* Transfection at eMedicine Dictionary
* Overview of transfection methods in Nature Methods 2, 875 - 883 (2005)
* Research in optical transfection at the University of St Andrews

Retrieved from "http://en.wikipedia.org/"
All text is available under the terms of the GNU Free Documentation License

Index

Scientific Library - Scientificlib.com