Transfection

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Transfection is the process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells.[1] 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 (carcinogenesis) 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 (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, 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[edit]

The meaning of the term has evolved.[2] The original meaning of transfection was "infection by transformation," i.e., introduction of 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[edit]

There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, cell squeezing, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers.

Chemical-based transfection[edit]

Chemical-based transfection can be divided into several kinds: cyclodextrin,[3] polymers,[4] liposomes, or nanoparticles [5] (with or without chemical or viral functionalization. See below).

Non chemical methods[edit]

Particle-based methods[edit]

Viral methods[edit]

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. [16]

Other (and hybrid) methods[edit]

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, [17] heat shock.

Stable and transient transfection[edit]

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.[18]

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.

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[edit]

Main article: 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 (to replace the "knock-down" experiments, to study the expression of proteins, i.e. of Endothelin-1[19]) 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[edit]

References[edit]

  1. ^ http://www.promega.com/paguide/chap12.htm
  2. ^ "Transfection" at Dorland's Medical Dictionary
  3. ^ Menuel S, Fontanay S, Clarot I, Duval R.E, Diez L, Marsura A (2008). "Synthesis and Complexation Ability of a Novel Bis- (guanidinium)-tetrakis-(β-cyclodextrin) Dendrimeric Tetrapod as a Potential Gene Delivery (DNA and siRNA) System. Study of Cellular siRNA Transfection". Bioconjugate Chem. 19 (12): 2357–2362. doi:10.1021/bc800193p. PMID 19053312. 
  4. ^ Fischer D, von Harpe A, Kunath K, Petersen H, Li YX, Kissel T (2002). "Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes". Bioconjugate Chem. 13 (5): 1124–1133. doi:10.1021/bc025550w. 
  5. ^ http://www.transfection.ws/nanoparticle_based_transfection_reagents
  6. ^ Graham FL, van der Eb AJ (1973). "A new technique for the assay of infectivity of human adenovirus 5 DNA". Virology 52 (2): 456–67. doi:10.1016/0042-6822(73)90341-3. PMID 4705382. 
  7. ^ Bacchetti S, Graham F (1977). "Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA". Proc Natl Acad Sci USA 74 (4): 1590–4. doi:10.1073/pnas.74.4.1590. PMC 430836. PMID 193108. 
  8. ^ Kriegler, Michael (1991). Transfer and Expression: A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96–97. ISBN 0716770040. 
  9. ^ Biological Sciences - Applied Biological Sciences: Armon Sharei, Janet Zoldan, Andrea Adamo, Woo Young Sim, Nahyun Cho, Emily Jackson, Shirley Mao, Sabine Schneider, Min-Joon Han, Abigail Lytton-Jean, Pamela A. Basto, Siddharth Jhunjhunwala, Jungmin Lee, Daniel A. Heller, Jeon Woong Kang, George C. Hartoularos, Kwang-Soo Kim, Daniel G. Anderson, Robert Langer, and Klavs F. Jensen A vector-free microfluidic platform for intracellular delivery PNAS 2013 ; published ahead of print January 22, 2013, doi:10.1073/pnas.1218705110.
  10. ^ M. Tsukakoshi, S. Kurata, Y. Nomiya, et al., "A Novel Method of DNA Transfection by Laser Microbeam Cell Surgery". Applied Physics B-Photophysics and Laser Chemistry. 35(3): 135–140 (1984)
  11. ^ Zhang, G., Budker, V. and Wolff, J.A. (1999) High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther., 10, 1735–1737.
  12. ^ Zhang, G., Vargo, D., Budker, V., N., A., Knechtle, S. and Wolff, J.A. (1997) Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Hum. Gene Ther., 8, 1763–1772.
  13. ^ Bell, J.B., Podetz-Pedersen, K., Aronovich, E.L., Belur, L.R., McIvor, R.S. and Hackett, P.B. (2007) Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection. Nat. Protocols, 2, 3153–3165.
  14. ^ http://www.ozbiosciences.com/magnetofection.html
  15. ^ "Transfection". 
  16. ^ "Transfection". 
  17. ^ Schnoor, Michael (March 2009). "Efficient Non-Viral Transfection of THP-1 Cells". Journal of Immunological Methods 344: 109–115. 
  18. ^ Nucleic Acids Res. 2002 January 15; 30(2): e9. "High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells"
  19. ^ Mawji et al used this technique to evaluate the kinetics of Endothelin-1 RNA decay in primary endothelial cells: Mawji et al.  "RNA transfection is a versatile tool to investigate endothelin-1 posttranscriptional regulation."  Exp Biol Med.  2006 Jun;231(6):704-8.  PMID 16740984

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