Transfection is the process of introducing nucleic acids into cells by non-viral methods [http://www.promega.com/guides/transfxn_guide/transfxn.pdf] . The term transformation is preferred to describe non-viral
DNAtransfer in bacteriaand non-animal eukaryotic cells such as fungi, algae and plants.
animal cells typically involves opening transientpores or 'holes' in the cell plasma membrane, to allow the uptake of material. Geneticmaterial (such as supercoiled plasmid DNA or siRNAconstructs), or even proteins such as antibodies, may be transfected. In addition to electroporation, transfection can be carried out by mixing a cationic lipidwith the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.
The original meaning of
transfectionwas 'infection by transformation', "i.e." introduction of DNA (or RNA) from an eukaryote virus or bacteriophageinto 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.
There are various methods of introducing foreign
DNAinto 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 (and least reliable) methods is transfection by
calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973 [cite journal |author=Graham FL, van der Eb AJ |title=A new technique for the assay of infectivity of human adenovirus 5 DNA |journal=Virology |volume=52 |issue=2 |pages=456–67 |year=1973 |pmid=4705382 |doi=10.1016/0042-6822(73)90341-3] (see also [cite journal |author=Bacchetti S, Graham F |title=Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA |journal=Proc Natl Acad Sci U S A |volume=74 |issue=4 |pages=1590–4 |year=1977 |pmid=193108 |doi=10.1073/pnas.74.4.1590] ). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloridesolution 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
polycationand 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 nanoparticleof an inertsolid (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.
Other methods of transfection include
nucleofection, electroporation, heat shock, magnetofectionand proprietary transfection reagents such as Lipofectamine, DojindoHilymax, Fugene, jetPEI, Effectene or DreamFect.
table and transient transfection
For most applications of transfection, it is sufficient if the transfected gene is only transiently expressed. Since the DNA introduced in the transfection process is usually not inserted into the nuclear genome, the foreign DNA is lost at the later stage when the cells undergo
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, another gene is co-transfected, which gives the cell some
selectionadvantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. If the toxin, towards which the co-transfected gene offers resistance, is then added to the cell culture, only those few cells with the foreign genes inserted into their genome will be able to proliferate, while other cells will die. After applying this selectionpressure 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 neomycinresistant gene.
* [http://www.nature.com/nmeth/journal/v2/n11/full/nmeth1105-875.html Overview of transfection methods] in "Nature Methods" 2, 875 - 883 (2005)
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