Transfection is the process of deliberately introducing nucleic acids into cells. The
term is often used for non-viral methods 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, including
plant cells. In animal cells, transfection is the preferred term as transformation is
also used to refer to progression to a cancerous state in these cells. Transduction is often
used to describe virus-mediated DNA transfer. The word transfection is a blend of trans-
and infection. Genetic material, 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, by cell squeezing 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. The original
meaning of transfection was "infection by transformation," i.e., introduction of DNA
from a prokaryote-infecting virus or bacteriophage into cells, resulting in an infection. Because
the term transformation had another sense in animal cell biology, 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, other on chemical materials or biological particles that are used as carriers.
Chemical-based transfection Chemical-based transfection can be divided
into several kinds: cyclodextrin, polymers, liposomes, or nanoparticles .
One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and
A. J. van der Eb in 1973. HEPES-buffered saline solution 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. By a process not entirely
understood, the cells take up some of the precipitate, and with it, the DNA. This process
has been a preferred method of identifying many oncogenes.
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 liposomes, because the cells are more sensitive. See lipofection for more details.
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. Non-chemical methods
Electroporation is a popular method, where transient increase in the permeability of
cell membrane is achieved when the cells are exposed to short pulses of an intense electric
field. Cell squeezing is a method invented in 2013
by Armon Sharei, Robert Langer and Klavs Jensen at MIT. It enables delivery of molecules into
cells by a gentle squeezing of the cell membrane. It is a high throughput vector-free microfluidic
platform for intracellular delivery. It eliminates the possibility of toxicity or off-target
effects as it does not rely on exogenous materials or electrical fields.
Sonoporation uses high-intensity ultrasound to induce pore formation in cell membranes.
This pore formation is attributed mainly to the cavitation of gas bubbles interacting
with nearby cell membranes since is enhanced by the addition of ultrasound contrast agent,
a source of cavitation nuclei. Optical transfection is a method where a tiny
hole is transiently generated in the plasma membrane of a cell using a highly focused
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. In
this technique, one cell at a time is treated, making it particularly useful for single cell
analysis. Protoplast fusion is a technique in which
transformed bacterial cells are treated with lysozyme in order to remove the cell wall.
Following this, fusogenic agents are used in order to fuse the protoplast carrying the
gene of interest with the target recipient cell. A major disadvantage of this method
is that bacterial components are non-specifically introduced into the target cell as well.
Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is
inserted into a cell. This approach can also be implemented with arrays of nanofibers that
are introduced into large numbers of cells and intact tissue.
Hydrodynamic delivery In mice and rats, but to a lesser extent in larger animals, DNA
most often in plasmids, including transposons, can be delivered to the liver using hydrodynamic
injection that involves infusion of a relatively large volume in the blood in less than 10
seconds; nearly all of the DNA is expressed in the liver by this procedure.
Particle-based methods A direct approach to transfection is the gene
gun, where the DNA is coupled to a nanoparticle of an inert solid 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. Impalefection is carried out by impaling cells
by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers
or silicon nanowires which have been functionalized with plasmid DNA.
Another particle-based method of transfection is known as particle bombardment. The nucleic
acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles.
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. 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. Other 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, heat shock. Stable and transient transfection
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 nuclear antigen 1 or the SV40 large-T
antigen, allow episomal amplification of plasmids containing the viral EBV or SV40 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 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 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 resistance gene.
RNA transfection
RNA can also be transfected into cells to transiently express its coded protein, or
to study RNA decay kinetics. The latter application is referred as siRNA transfection or RNA silencing,
and has become a major application in research 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 Impalefection
References
External links Transfection at the US National Library of
Medicine Medical Subject Headings Biology Research Resource – Articles and
Forums about Transfection Transfection at eMedicine Dictionary
Overview of transfection methods in Nature Methods 2, 875–883
Research in optical transfection at the University of St Andrews