Difference between revisions of "Transfection" - New World Encyclopedia

From New World Encyclopedia
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[[Image:genegun.jpg|thumb|PSD-1000/He Particle Delivery System. The ''gene gun'' or the Biolistic Particle Delivery System, originally designed for [[plant]] [[transformation (genetics)|transformation]], is a device for injecting cells with [[genetics|genetic]] information]].
 
[[Image:genegun.jpg|thumb|PSD-1000/He Particle Delivery System. The ''gene gun'' or the Biolistic Particle Delivery System, originally designed for [[plant]] [[transformation (genetics)|transformation]], is a device for injecting cells with [[genetics|genetic]] information]].
'''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.  
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'''Transfection''' is the process of artificially introducing [[nucleic acid]]s ([[DNA]] or [[RNA]]) into [[cell (biology)|cells]], utilizing means other than through [[virus|viral]] infection. Such introductions of foreign DNA can result in a change of the properties of the cell. Classically the term was defined more narrowly as the process of infecting a bacterial cell with viral DNA, or 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). Now it includes any artificial introduction of foreign DNA into a cell. 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.
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:artificial infection of bacterial cells by uptake of viral nucleic acid, resulting in the production of mature virus particles.
  
 
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.
 
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.
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stable integration of the DNA into the recipient genome may be called stable transfection.
  
 
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.   
 
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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http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?pg=/ppdocs/us/common/dorlands/dorland/eight/000110186.htm
 
http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?pg=/ppdocs/us/common/dorlands/dorland/eight/000110186.htm
 
* Dorland, W. A. N. 2007. [http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?pg=/ppdocs/us/common/dorlands/dorland/five/000059130.htm Ligament]. ''Dorland's Illustrated Medical Dictionary''. Edinburgh: Elsevier Saunders. ISBN 9781416023647.
 
* Dorland, W. A. N. 2007. [http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?pg=/ppdocs/us/common/dorlands/dorland/five/000059130.htm Ligament]. ''Dorland's Illustrated Medical Dictionary''. Edinburgh: Elsevier Saunders. ISBN 9781416023647.
Originally: artificial infection of bacterial cells by uptake of viral nucleic acid, resulting in the production of mature virus particles. Now, it includes any means of artificial introduction of foreign DNA into cultured eukaryotic cells; stable integration of the DNA into the recipient genome may be called stable transfection.
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Originally: artificial infection of bacterial cells by uptake of viral nucleic acid, resulting in the production of mature virus particles. Now, it includes any means of artificial introduction of foreign DNA into cultured eukaryotic cells;  
  
  
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Promega. n.d.  
 
Promega. n.d.  
<ref>http://www.promega.com/guides/transfxn_guide/transfxn.pdf Transfection
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http://www.promega.com/guides/transfxn_guide/transfxn.pdf Transfection
  
  
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* 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     
 
* 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     
  
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Transfection' vs 'Transformation': Defining Terms
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Author: Seeber F.
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Source: Parasitology Today, Volume 16, Number 9, 1 September 2000 , pp. 404-404(1)
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http://www.ncbi.nlm.nih.gov/pubmed/10951602
  
  

Revision as of 13:18, 21 February 2009

PSD-1000/He Particle Delivery System. The gene gun or the Biolistic Particle Delivery System, originally designed for plant transformation, is a device for injecting cells with genetic information

.

Transfection is the process of artificially introducing nucleic acids (DNA or RNA) into cells, utilizing means other than through viral infection. Such introductions of foreign DNA can result in a change of the properties of the cell. Classically the term was defined more narrowly as the process of infecting a bacterial cell with viral DNA, or 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). Now it includes any artificial introduction of foreign DNA into a cell. The term transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae, and plants.

artificial infection of bacterial cells by uptake of viral nucleic acid, resulting in the production of mature virus particles.

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.

stable integration of the DNA into the recipient genome may be called stable transfection.

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

introduce nucleic acids into cells

gene regulation analysis of expresson and function of proteins within eukarotic cells, such as mammalinan cells, production of transgenic organsims gene therapy strategies


Terminology

The meaning of the term transfection has evolved.Cite error: Closing </ref> missing for <ref> tag

Separate terms are used for genetic alterations resulting from introduction of DNA by viruses ("transduction") or by cell-cell contact between bacteria ("conjugation"). Transformation of eukaryotic cells in tissue culture is usually called transfection.

This

process is distinct from “infection”, which is a viral method of nucleic acid introduction into cells.


Promega. n.d. http://www.promega.com/guides/transfxn_guide/transfxn.pdf Transfection


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.


The

techniques developed for gene transfer can be broadly classified as either chemical reagents or physical methods. Chemical Reagents DEAE-dextran was one of the first chemical reagents used for transfer of nucleic acids into cultured mammalian cells (1,9). Calcium phosphate co

physical Direct microinjection into cultured cells or nuclei is an effective, although Electroporation was first reported for gene transfer studies in 1982 (4). This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell Another physical method of gene delivery is biolistic particle delivery. This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells

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.

An example of such a combination is the marker gene for neomycin phosphotransferase with the drug Geneticin® (8).

See also

  • Protofection
  • Transformation
  • Transduction
  • Cationic liposome
  • Nucleofection

References
ISBN links support NWE through referral fees

  • Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1989. Molecular Biology of the Cell, 2nd edition. New York: Garland Publishing. ISBN 0824036956.
  • 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


Transfection' vs 'Transformation': Defining Terms Author: Seeber F. Source: Parasitology Today, Volume 16, Number 9, 1 September 2000 , pp. 404-404(1) http://www.ncbi.nlm.nih.gov/pubmed/10951602


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