Transfection

Transfection is the process of deliberately introducing naked or purified nucleic acids by non-viral methods into eukaryotic cells.[1][2] 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 and 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 gene transfer into eukaryotic cells.[2][3]

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 (i.e. tricalcium 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.[4] 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

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. Gene delivery is, for example, one of the steps necessary for gene therapy and the genetic modification of crops. There are many different methods of gene delivery developed for a various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into two categories, non-viral and viral.[5]

Non-viral methods include physical methods such as electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, and sonication and chemical, such as lipofection. It can also include the use of polymeric gene carriers (polyplexes).[6]

Virus mediated gene delivery utilizes the ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a replication-deficient viral particle. Viruses used to date include retrovirus, adenovirus, adeno-associated virus and herpes simplex virus. However, there are drawbacks to using viruses to deliver genes into cells. Viruses can only deliver very small pieces of DNA into the cells, it is labor-intensive and there are risks of random insertion sites, cytopathic effects and mutagenesis.

Nonviral methods

Chemical-based transfection

Chemical-based transfection can be divided into several kinds: cyclodextrin,[7] polymers,[8] liposomes, or nanoparticles[9] (with or without chemical or viral functionalization. See below).

Non-chemical methods

Electroporator with square wave and exponential decay waveforms for in vitro, in vivo, adherent cell and 96 well electroporation applications. Manufactured by BTX Harvard Apparatus, Holliston MA USA.

Particle-based methods

Other (and hybrid) 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,[21] heat shock.

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.[2] Lentiviral vectors are also helpful due to their ability to transduce cells not currently undergoing mitosis.

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 (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.[22]

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

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[23]) 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

References

  1. Transfection at the US National Library of Medicine Medical Subject Headings (MeSH)
  2. 1 2 3 4 "Transfection". Protocols and Applications Guide. Promega.
  3. Transduction, Genetic at the US National Library of Medicine Medical Subject Headings (MeSH)
  4. "Transfection" at Dorland's Medical Dictionary
  5. Kamimura K, Suda T, Zhang G, et al. (2011). "Advances in Gene Delivery Systems". Pharm Med. 25 (5): 293–306. doi:10.2165/11594020-000000000-00000.
  6. Saul JM, Linnes MP, Ratner BD, Giachelli CM, Pun SH (November 2007). "Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression". Biomaterials. 28 (31): 4705–16. doi:10.1016/j.biomaterials.2007.07.026. PMID 17675152.
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  8. 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): 1124–33. doi:10.1021/bc025550w.
  9. "Nanoparticle Based Transfection Reagents". Biology Transfection Research Resource. Transfection.ws.
  10. 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.
  11. 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 430836Freely accessible. PMID 193108.
  12. Kriegler, Michael (1991). Transfer and Expression: A Laboratory Manual. W. H. Freeman. pp. 96–97. ISBN 0716770040.
  13. Felgner PL, Gadek TR, Holm M, et al. (November 1987). "Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure". Proc. Natl. Acad. Sci. U.S.A. 84 (21): 7413–7. doi:10.1073/pnas.84.21.7413. PMC 299306Freely accessible. PMID 2823261.
  14. Felgner JH, Kumar R, Sridhar CN, et al. (January 1994). "Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations". J. Biol. Chem. 269 (4): 2550–61. PMID 8300583.
  15. Sharei A, Zoldan J, Adamo A, Sim WY, Cho N, Jackson E, Mao S, Schneider S, Han MJ, Lytton-Jean A, Basto PA, Jhunjhunwala S, Lee J, Heller DA, Kang JW, Hartoularos GC, Kim KS, Anderson DG, Langer R, Jensen KF (February 2013). "A vector-free microfluidic platform for intracellular delivery". Proc. Natl. Acad. Sci. U.S.A. 110 (6): 2082–7. doi:10.1073/pnas.1218705110. PMC 3568376Freely accessible. PMID 23341631.
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  17. Zhang G, Budker V, Wolff JA (July 1999). "High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA". Hum. Gene Ther. 10 (10): 1735–7. doi:10.1089/10430349950017734. PMID 10428218.
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  19. Bell JB, Podetz-Pedersen KM, Aronovich EL, Belur LR, McIvor RS, Hackett PB (2007). "Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection". Nat Protoc. 2 (12): 3153–65. doi:10.1038/nprot.2007.471. PMC 2548418Freely accessible. PMID 18079715.
  20. "Magnetofection — Magnetic assisted transfection & transduction". OzBiosciences—The art of delivery systems.
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  22. Durocher Y, Perret S, Kamen A (January 2002). "High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells". Nucleic Acids Res. 30 (2): E9. doi:10.1093/nar/30.2.e9. PMC 99848Freely accessible. PMID 11788735.
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Further reading

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