Practice and methods: gene transfection

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pguo
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Practice and methods: gene transfection

Source: http://www.e-bioinfo.com
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 such as fungi, algae and plants.

Transfection of animal cells typically involves opening transient pores or 'holes' in the cell plasma 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. In addition to electroporation, transfection can be carried out by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.

The original meaning of transfection was 'infection by transformation', i.e. introduction of DNA (or RNA) from an eukaryote 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 (and least reliable) methods is transfection by calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[2] (see also [3]). 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.

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

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 later stage 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 resistant gene.

More protocols on gene transfection and molecular biology:
http://www.e-bioinfo.com/bioprotocols.html

Shubhangi
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Hi

Hi
Just to add more to above general information:
Transformation and transfection efficiency is very important to get large number of clones. Also there should be clear differentiation between transfected and native-non transfected cells. This is possible because of presence some or other kind of specific gene in transfected vector ( DNA fragment that carries gene of interest).
This specific gene is ususally antibiotic resistant gene. In some cases it not differentiates transfected and non transfected cells but also helps in production of gene of interest. As you increase concentration of antibiotic in medium more production of desired protein occurs.