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Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Transfection typically involves opening transient "holes" or gates in cells to allow the entry of extracellular molecules, typically supercoiled plasmid DNA, but also siRNA, among others. Transfection differs from transformation since the DNA is not generally incorporated into the cell's genome, it is only transiently expressed.
Methods of transfection
There are various methods of introducing foreign DNA into a cell. One of the cheapest (and least reliable) ones is transfection by calcium phosphate, originally discovered by S. Bacchetti and F. L. Graham in 1977. 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 calcium 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 yet, the cells will take up some of the precipitate, and with it, the DNA. Electroporation, heat shock, and proprietary transfection reagents such as Fugene are other methods.
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, as the cells are more sensitive.
A very direct approach is the so-called "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.
Stable 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 latest when the cells undergo mitosis. 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 selection advantage, 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 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 resistance gene.
References
Bacchetti and Graham in PNAS 74, p. 1590-94, available from the NCBI: *
External links
- Transfection Protocols : Contains various protocols for transfection, clone picking and screening. Subcategories include: Electroporation, Reporter Analysis, and Stable Transfections.
"Transfection"