Difference between revisions of "Transfection" - New World Encyclopedia
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| − | '''Transfection''' is the process of introducing nucleic | + | '''Transfection''' is the process of introducing [[nucleic acid]]s ([[DNA]] or [[RNA]]) into [[cell (biology)|cells]] by means other than through [[virus|viral]] infection. Such introductions can result in a change of the properties of the cell. Classically the term was defined more narrowly as the process of infecting a cell with viral nucleic acid, either isolated from a eukaryote virus or from a [[bacteriophage]] (a virus that infects bacteria). The term [[transformation (genetics)|transformation]] is preferred to describe non-viral [[DNA]] transfer in [[bacteria]] and non-animal eukaryotic cells such as [[fungus|fungi]], [[alga]]e, and [[plant]]s. |
| + | |||
| + | The introduced nucleic acid may be transient, such that it does not replicate, or it may be stable and insert into the genome and replicate when the host genome replicates. | ||
| + | |||
| + | There are many methods employed in performing transfection. Transfection of [[animal cell]]s typically involves opening transient pores or “holes” in the cell [[plasma membrane]], to allow the uptake of material. [[Genetics|Genetic]] material (such as [[DNA supercoil|supercoiled plasmid DNA]] or [[siRNA]] constructs), or even sections of DNA coding for [[protein]]s such as [[antibody|antibodies]], may be transfected. In addition to [[electroporation]], transfection can be carried out using [[calcium phosphate]], or by mixing a [[cationic]] [[lipid]] with the material to produce [[liposome]]s, which fuse with the cell plasma membrane and deposit their cargo inside. | ||
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| + | ? insulin production | ||
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==Terminology== | ==Terminology== | ||
| − | The meaning of the term has evolved.<ref>{{DorlandsDict|eight/000110186|Transfection}}</ref> | + | The meaning of the term transfection has evolved.<ref>{{DorlandsDict|eight/000110186|Transfection}}</ref> |
| + | |||
| + | 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 nucleic acids by non-viral methods. | ||
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| + | <ref>http://www.promega.com/guides/transfxn_guide/transfxn.pdf</ref>. | ||
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==Methods== | ==Methods== | ||
| − | There are various methods of introducing foreign [[DNA]] into a eukaryotic [[cell (biology)|cell]]. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles. | + | There are various methods of introducing foreign [[DNA]] into a eukaryotic [[cell (biology)|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 | + | 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 and reported on in 1973 (Graham and Eb 1973; Bacchetti and Graham 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 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 [[ | + | 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 [[liposome]]s, in other words, 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|DEAE-dextran]] or [[polyethylenimine]]. The negatively charged DNA binds to the [[polycation]] and the complex is taken up by the cell via [[endocytosis]]. | Another method is the use of cationic polymers such as [[DEAE|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 [[cell nucleus|nucleus]]. DNA can | + | 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 [[cell nucleus|nucleus]]. |
| + | |||
| + | Of course, DNA also can be introduced into cells using [[virus]]es as a carrier; in such cases, the technique is called viral [[transduction (genetics)|transduction]], and, the cells are said to be transduced. | ||
| − | Other methods of transfection include [[nucleofection]], [[electroporation]], [[sonoporation]], [[heat shock]], [[magnetofection]] and proprietary transfection reagents such as [[Lipofectamine]], [[Dojindo]] Hilymax, Fugene, jetPEI, Effectene or DreamFect. | + | Other methods of transfection include [[nucleofection]], [[electroporation]], [[sonoporation]], [[heat shock]], [[magnetofection]], and proprietary transfection reagents such as [[Lipofectamine]], [[Dojindo]] Hilymax, Fugene, jetPEI, Effectene, or DreamFect. |
==Stable and transient transfection== | ==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]]. | 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. | + | 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 [[cell proliferation|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. | 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. | ||
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==References== | ==References== | ||
| − | + | ||
| + | Alberts | ||
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| + | * Graham, F. L., and A. J. van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456–67. PMID 4705382. | ||
| + | |doi=10.1016/0042-6822(73)90341-3}} | ||
| + | |||
| + | * Bacchetti, S. and F. L. Graham. 1977. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proc Natl Acad Sci U S A 74(4): 1590–4. PMID 193108 | ||
| + | |||
| + | . | ||
| + | |||
==External links== | ==External links== | ||
Revision as of 12:31, 21 February 2009
Transfection is the process of introducing nucleic acids (DNA or RNA) into cells by means other than through viral infection. Such introductions can result in a change of the properties of the cell. Classically the term was defined more narrowly as the process of infecting a cell with viral nucleic acid, either isolated from a eukaryote virus or from a bacteriophage (a virus that infects bacteria). The term transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae, and plants.
The introduced nucleic acid may be transient, such that it does not replicate, or it may be stable and insert into the genome and replicate when the host genome replicates.
There are many methods employed in performing transfection. 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 sections of DNA coding for proteins such as antibodies, may be transfected. In addition to electroporation, transfection can be carried out using calcium phosphate, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside.
? insulin production
Terminology
The meaning of the term transfection has evolved.[1]
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 nucleic acids by non-viral methods.
[2].
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 and reported on in 1973 (Graham and Eb 1973; Bacchetti and Graham 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 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, in other words, 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.
Of course, DNA also can 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, sonoporation, 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.
See also
- Protofection
- Transformation
- Transduction
- Cationic liposome
- Nucleofection
ReferencesISBN links support NWE through referral fees
Alberts
- Graham, F. L., and A. J. van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456–67. PMID 4705382.
|doi=10.1016/0042-6822(73)90341-3}}
- Bacchetti, S. and F. L. Graham. 1977. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proc Natl Acad Sci U S A 74(4): 1590–4. PMID 193108
.
External links
- MeSH Transfection
- Template:EMedicineDictionary
- Overview of transfection methods in Nature Methods 2, 875 - 883 (2005)
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Credits
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- ↑ Template:DorlandsDict
- ↑ http://www.promega.com/guides/transfxn_guide/transfxn.pdf
