Transfection
Encyclopedia
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably 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 and plant cells - a distinctive sense of transformation refers to spontaneous genetic modifications (mutation
s to cancerous cells (Carcinogenesis
), or under stress (UV irradiation)). "Transduction
" is often used to describe virus-mediated DNA transfer.
The word transfection is a blend
of trans- and infection.
Genetic
material (such as supercoiled plasmid DNA
or siRNA
constructs), or even protein
s 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
, 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.
-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.
into a eukaryotic cell
: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers.
, polymers, liposomes, or nanoparticles (with or without chemical or viral functionalization. See below).
es as a carrier. In such cases, the technique is called viral transduction
, and the cells are said to be transduced. This can be done using insect cells.
, heat shock
.
or degraded.
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, 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.
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.
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.
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
" is more often used to describe non-viral DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
transfer in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, non-animal eukaryotic
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
cells and plant cells - a distinctive sense of transformation refers to spontaneous genetic modifications (mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s to cancerous cells (Carcinogenesis
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
), or under stress (UV irradiation)). "Transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
" is often used to describe virus-mediated DNA transfer.
The word transfection is a blend
Blend
In linguistics, a blend is a word formed from parts of two or more other words. These parts are sometimes, but not always, morphemes.-Linguistics:...
of trans- and infection.
Genetic
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
material (such as supercoiled plasmid DNA
DNA supercoil
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...
or siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
constructs), or even protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s 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
Calcium phosphate
Calcium phosphate is the name given to a family of minerals containing calcium ions together with orthophosphates , metaphosphates or pyrophosphates and occasionally hydrogen or hydroxide ions ....
, by electroporation
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
, or by mixing a cationic lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
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 (or RNA) from a prokaryoteProkaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...
-infecting virus or bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
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 DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
into a eukaryotic cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers.
Chemical-based transfection
Chemical-based transfection can be divided into several kinds: cyclodextrinCyclodextrin
Cyclodextrins are a family of compounds made up of sugar molecules bound together in a ring ....
, polymers, liposomes, or nanoparticles (with or without chemical or viral functionalization. See below).
- One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973 (see also ). HEPESHEPESHEPES is a zwitterionic organic chemical buffering agent; one of the twelve Good's buffers...
-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chlorideCalcium chlorideCalcium chloride, CaCl2, is a salt of calcium and chlorine. It behaves as a typical ionic halide, and is solid at room temperature. Common applications include brine for refrigeration plants, ice and dust control on roads, and desiccation...
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 membraneCell membraneThe cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
, releasing the DNA into the cell. For eukaryotic cells, transfection is better achieved using cationic liposomes (or mixtures), because the cells are more sensitive. See lipofectionLipofectionLipofection is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged lipid to form an aggregate with the...
for more details.
- Another method is the use of cationic polymers such as DEAE-dextranDEAEDiethylaminoethyl cellulose is a positively charged resin used in ion exchange chromatography, a type of column chromatography, used in protein and nucleic acid purification/separation...
or polyethyleniminePolyethylenimineLinear polyethyleneimines contain all secondary amines, in contrast to branched PEIs which contain primary, secondary and tertiary amino groups. The linear PEIs are solids at room temperature where branched PEIs are liquids at all molecular weights. Linear polyethyleneimines are soluble in hot...
. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosisEndocytosisEndocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
.
Non chemical methods
- ElectroporationElectroporationElectroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
is a popular method, although requiring an instrument and affecting the viability of many cell types, that also creates micro-sized holes transiently in the plasma membrane of cells under an electric discharge.
- Similarly, transfection applying sonic forces to cells, referred as Sono-porationSonoporationSonoporation, or cellular sonication, is the use of sound for modifying the permeability of the cell plasma membrane. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process...
.
- Optical transfectionOptical transfectionOptical transfection is the process of introducing nucleic acids into cells using light. Typically, a laser is focussed to a diffraction limited spot using a high numerical aperture microscope objective...
is a method where a tiny (~1 µm diameter) 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.
- Gene electrotransferGene electrotransferGene electrotransfer is a versatile biotechnology technique that enables transfer of genetic material into prokaryotic or eukaryotic cells. It is based on a physical method named electroporation, where transient increase in the permeability of cell membrane is achieved when submitted to short and...
is a technique that enables transfer of genetic material into prokaryotic or eukaryotic cells. It is based on a physical method named electroporationElectroporationElectroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
, where transient increase in the permeability of cell membrane is achieved when submitted to short and intense electric pulses.
- ImpalefectionImpalefectionImpalefection is a method of gene delivery using nanomaterials, such as carbon nanofibers, carbon nanotubes, nanowires Ref.1. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. Plasmid DNA containing the gene, intended for intracellular delivery, is attached to...
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 gunGene gunA gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA...
, where the DNA is coupled to a nanoparticleNanoparticleIn nanotechnology, a particle is defined as a small object that behaves as a whole unit in terms of its transport and properties. Particles are further classified according to size : in terms of diameter, coarse particles cover a range between 10,000 and 2,500 nanometers. Fine particles are sized...
of an inertInert-Chemistry:In chemistry, the term inert is used to describe a substance that is not chemically reactive.The noble gases were previously known as inert gases because of their perceived lack of participation in any chemical reactions...
solid (commonly gold) which is then "shot" directly into the target cell's nucleusCell nucleusIn cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
.
- MagnetofectionMagnetofectionMagnetofection is a simple and highly efficient transfection method that uses magnetic fields to concentrate particles containing nucleic acid into the target cells...
, or Magnet assisted transfectionMagnet assisted transfectionMagnet Assisted Transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Therefore, nucleic acids are first associated with magnetic nanoparticles...
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.
- ImpalefectionImpalefectionImpalefection is a method of gene delivery using nanomaterials, such as carbon nanofibers, carbon nanotubes, nanowires Ref.1. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. Plasmid DNA containing the gene, intended for intracellular delivery, is attached to...
is carried out by impaling cells by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers or siliconSiliconSilicon is a chemical element with the symbol Si and atomic number 14. A tetravalent metalloid, it is less reactive than its chemical analog carbon, the nonmetal directly above it in the periodic table, but more reactive than germanium, the metalloid directly below it in the table...
nanowires which have been functionalized with plasmidPlasmidIn microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
.
Viral methods
DNA can also be introduced into cells using virusVirus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es as a carrier. In such cases, the technique is called viral transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
, and the cells are said to be transduced. This can be done using insect cells.
Other (and hybrid) methods
Other methods of transfection include nucleofectionNucleofection
Nucleofection refers to electroporation, a transfection method which enables transfer of nucleic acids such as DNA, RNA, Small interfering RNA into cells. Nucleofection, also referred to as Nucleofector Technology, was invented by the biotechnology company amaxa...
, heat shock
Heat shock
In biochemistry, heat shock is the effect of subjecting a cell to a higher temperature than that of the ideal body temperature of the organism from which the cell line was derived.-Heat shock response:...
.
Stable and transient transfection
For most 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 mitosisMitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
or degraded.
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, a marker gene
Marker gene
A marker gene is a gene used in molecular biology to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. There are two types of marker genes: a selectable marker and a marker for screening.-Selectable marker:...
is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...
. 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.
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
Neomycin
Neomycin is an aminoglycoside antibiotic that is found in many topical medications such as creams, ointments, and eyedrops. The discovery of Neomycin dates back to 1949. It was discovered in the lab of Selman Waksman, who was later awarded the Nobel Prize in Physiology and medicine in 1951...
resistance gene.
RNA transfection
RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. The later application is referred as siRNA transfection or RNA silencing, and has become a major application in research (to replace the "knock-down" experiments, to study the expression of proteins, i.e. of Endothelin-1 ) 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
- ProtofectionProtofectionProtofection is a term that refers to the transfection of foreign mitochondrial DNA to replace the original energy generators within cells. As mitochondria are damaged with age, this would be a method of rejuvenating them to original states....
- TransformationTransformation (genetics)In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
- TransductionTransduction (genetics)Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
- Cationic liposomeCationic liposome- life :Cationic liposomes are structures that are made of positively charged lipids and are increasingly being researched for use in gene therapy due to their favourable interactions with negatively charged DNA and cell membranes. Cationic liposomes are also known as cationic lipoplexes....
- NucleofectionNucleofectionNucleofection refers to electroporation, a transfection method which enables transfer of nucleic acids such as DNA, RNA, Small interfering RNA into cells. Nucleofection, also referred to as Nucleofector Technology, was invented by the biotechnology company amaxa...
- Magnet assisted transfectionMagnet assisted transfectionMagnet Assisted Transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Therefore, nucleic acids are first associated with magnetic nanoparticles...
- ImpalefectionImpalefectionImpalefection is a method of gene delivery using nanomaterials, such as carbon nanofibers, carbon nanotubes, nanowires Ref.1. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. Plasmid DNA containing the gene, intended for intracellular delivery, is attached to...