Transfection

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.
The word "transfection" is a portmanteau 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 (i.e. tricalcium phosphate), by electroporation, by cell squeezing or by mixing a cationic lipid with the material to produce liposomes that 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. The original meaning of transfection was "infection by transformation", i.e., introduction of genetic material, DNA or RNA, from a prokaryote-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, cell squeezing, nanoparticles, magnetofection); others rely on chemical materials or biological particles (viruses) that are used as carriers. Gene delivery is, for example, one of the steps necessary for gene therapy and the genetic modification of crops. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into two categories: non-viral and viral.
Non-viral methods include physical methods such as electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, and sonication and chemical, such as lipofection, which is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes).
Virus mediated gene delivery utilizes the ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a replication-deficient viral particle. Viruses used to date include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. However, there are drawbacks to using viruses to deliver genes into cells. Viruses can only deliver very small pieces of DNA into the cells, it is labor-intensive and there are risks of random insertion sites, cytopathic effects and mutagenesis.
Bacterial spheroplasts can transfect animal cells.
Nonviral methods.
Chemical-based transfection.
Chemical-based transfection can be divided into several kinds: cyclodextrin, polymers, liposomes, or nanoparticles (with or without chemical or viral functionalization. See below).
Other (and hybrid) methods.
Other methods of transfection include nucleofection, which has proved very efficient in transfection of the THP-1 cell line, creating a viable cell line that was able to be differentiated into mature macrophages, and heat shock.
Viral methods.
DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called transduction, and the cells are said to be transduced. Adenoviral vectors can be useful for viral transfection methods because they can transfer genes into a wide variety of human cells and have high transfer rates. Lentiviral vectors are also helpful due to their ability to transduce cells not currently undergoing mitosis.
Stable and transient transfection.
Stable and transient transfection differ in their long term effects on a cell; a stably-transfected cell will continuously express transfected DNA and pass it on to daughter cells, while a transiently-transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells.
For some 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. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution.
If it is desired that the transfected gene actually remain 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.
Common agents for selecting stable transfection are:
RNA transfection.
RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. RNA transfection is often used in primary cells that do not divide.
siRNAs can also be transfected to achieve RNA silencing (i.e. loss of RNA and protein from the targeted gene). This has become a major application in research to achieve "knock-down" of proteins of interests (e.g. Endothelin-1) with potential applications in gene therapy. Limitation of the silencing approach are the toxicity of the transfection for cells and potential "off-target" effects on the expression of other genes/proteins.