Transfection#Lipofection

The meaning of the term has evolved.{{DorlandsDict|eight/000110186|Transfection}} The original meaning of transfection was "infection by transformation", i.e., introduction of genetic material, DNA or RNA, from a prokaryote-infecting virus or bacteriophage into cells, resulting in an infection. For work with bacterial and archaeal cells transfection retains its original meaning as a special case of transformation. 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.{{fact|date=January 2022}}

There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, cell squeezing, nanoparticles, magnetofection); others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological.

Physical methods include electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, and sonication. Chemicals include methods such as lipofection, which is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes).{{cite journal | vauthors = Saul JM, Linnes MP, Ratner BD, Giachelli CM, Pun SH | title = Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression | journal = Biomaterials | volume = 28 | issue = 31 | pages = 4705–16 | date = November 2007 | pmid = 17675152 | doi = 10.1016/j.biomaterials.2007.07.026 }} Biological transfection is typically mediated by viruses, utilizing 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, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus.{{fact|date=January 2022}}

File:Gemini X2 generator.jpg

Physical methods are the conceptually simplest, using some physical means to force the transfected material into the target cell's nucleus. The most widely used physical method is electroporation, where short electrical pulses disrupt the cell membrane, allowing the transfected nucleic acids to enter the cell. Other physical methods use different means to poke holes in the cell membrane: Sonoporation uses high-intensity ultrasound (attributed mainly to the cavitation of gas bubbles interacting with nearby cell membranes), optical transfection uses a highly focused laser to form a ~1 μm diameter hole.{{cite journal |vauthors=Tsukakoshi M, Kurata S, Nomiya Y, etal |title=A Novel Method of DNA Transfection by Laser Microbeam Cell Surgery |journal=Applied Physics B: Photophysics and Laser Chemistry |volume=35 |issue=3 |pages=135–140 |year=1984 |doi=10.1007/BF00697702 |bibcode=1984ApPhB..35..135T|s2cid=123250337 }}

Several methods use tools that force the nucleic acid into the cell, namely: microinjection of nucleic acid with a fine needle; biolistic particle delivery, in which nucleic acid is attached to heavy metal particles (usually gold) and propelled into the cells at high speed;{{cite journal |vauthors=Mehier-Humbert S, Guy RH |title=Physical methods for gene transfer: improving the kinetics of gene delivery into cells |journal=Adv Drug Deliv Rev |volume=57 |issue=5 |pages=733–53 |date=April 2005 |pmid=15757758 |doi=10.1016/j.addr.2004.12.007 }} and magnetofection, where nucleic acids are attached to magnetic iron oxide particles and driven into the target cells by magnets.

Hydrodynamic delivery is a method used in mice and rats, in which nucleic acids can be delivered to the liver by injecting 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.{{cite book |vauthors=Suda T, Liu D |chapter=Hydrodynamic Delivery |title=Nonviral Vectors for Gene Therapy - Physical Methods and Medical Translation |series=Advances in Genetics |volume=89 |pages=89–111 |date=2015 |pmid=25620009 |doi=10.1016/bs.adgen.2014.10.002 |isbn=9780128022726 |chapter-url=}}

Chemical-based transfection can be divided into several kinds: cyclodextrin,{{cite journal | vauthors = Menuel S, Fontanay S, Clarot I, Duval RE, Diez L, Marsura A | title = Synthesis and complexation ability of a novel bis- (guanidinium)-tetrakis-(beta-cyclodextrin) dendrimeric tetrapod as a potential gene delivery (DNA and siRNA) system. Study of cellular siRNA transfection | journal = Bioconjugate Chemistry | volume = 19 | issue = 12 | pages = 2357–62 | date = December 2008 | pmid = 19053312 | doi = 10.1021/bc800193p }} polymers,{{cite journal | vauthors = Fischer D, von Harpe A, Kunath K, Petersen H, Li Y, Kissel T | title = 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 | journal = Bioconjugate Chemistry | volume = 13 | issue = 5 | pages = 1124–33 | year = 2002 | pmid = 12236795 | doi = 10.1021/bc025550w }} liposomes, or nanoparticles{{cite web |title=Nanoparticle Based Transfection Reagents |work=Biology Transfection Research Resource |publisher=Transfection.ws |url=http://www.transfection.ws/nanoparticle_based_transfection_reagents |access-date=30 September 2009 |archive-date=21 April 2013 |archive-url=https://archive.today/20130421222116/http://www.transfection.ws/nanoparticle_based_transfection_reagents |url-status=dead }} (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{{cite journal | vauthors = Graham FL, van der Eb AJ | title = A new technique for the assay of infectivity of human adenovirus 5 DNA | journal = Virology | volume = 52 | issue = 2 | pages = 456–67 | date = April 1973 | pmid = 4705382 | doi = 10.1016/0042-6822(73)90341-3 }} (see also{{cite journal | vauthors = Bacchetti S, Graham FL | title = Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 74 | issue = 4 | pages = 1590–4 | date = April 1977 | pmid = 193108 | pmc = 430836 | doi = 10.1073/pnas.74.4.1590 | bibcode = 1977PNAS...74.1590B | doi-access = free }}). 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. This process has been a preferred method of identifying many oncogenes.{{cite book | last = Kriegler | first = Michael | name-list-style = vanc | title = Transfer and Expression: A Laboratory Manual | year = 1991 | publisher = W. H. Freeman | isbn = 978-0-7167-7004-6 | pages = 96–97 }}
• Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.
• {{anchor|Lipofection}}Lipofection (or liposome transfection) 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.{{cite journal | vauthors = Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M | title = Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 84 | issue = 21 | pages = 7413–7 | date = November 1987 | pmid = 2823261 | pmc = 299306 | doi = 10.1073/pnas.84.21.7413 | bibcode = 1987PNAS...84.7413F | doi-access = free }} Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material.{{cite journal | vauthors = Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL | title = Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations | journal = The Journal of Biological Chemistry | volume = 269 | issue = 4 | pages = 2550–61 | date = January 1994 | doi = 10.1016/S0021-9258(17)41980-6 | pmid = 8300583 | doi-access = free }} This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, calcium phosphate, and electroporation. The efficiency of lipofection can be improved by treating transfected cells with a mild heat shock.{{cite journal | vauthors = Pipes BL, Vasanwala FH, Tsang TC, Zhang T, Luo P, Harris DT | title = Brief heat shock increases stable integration of lipid-mediated DNA transfections | journal = BioTechniques | volume = 38 | issue = 1 | pages = 48–52 | date = January 2005 | pmid = 15679084 | url = http://www.biotechniques.com/BiotechniquesJournal/2005/January/Brief-heat-shock-increases-stable-integration-of-lipid-mediated-DNA-transfections/biotechniques-45348.html | doi = 10.2144/05381bm05 | doi-access = free }}{{Dead link|date=April 2024 |bot=InternetArchiveBot |fix-attempted=yes }}
• Fugene is a series of widely used proprietary non-liposomal transfection reagents capable of directly transfecting a wide variety of cells with high efficiency and low toxicity.{{cite journal | vauthors = Jacobsen LB, Calvin SA, Colvin KE, Wright M | title = FuGENE 6 Transfection Reagent: the gentle power | journal = Methods | volume = 33 | issue = 2 | pages = 104–12 | date = June 2004 | pmid = 15121164 | doi = 10.1016/j.ymeth.2003.11.002 | series = Transfection of Mammalian Cells }}{{cite journal | vauthors = Hellgren I, Drvota V, Pieper R, Enoksson S, Blomberg P, Islam KB, Sylvén C | title = Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies | language = en | journal = Cellular and Molecular Life Sciences | volume = 57 | issue = 8–9 | pages = 1326–33 | date = August 2000 | pmid = 11028922 | doi = 10.1007/PL00000769 | s2cid = 27916034 | pmc = 11146917 }}{{cite book | first1 = Uma | last1 = Lakshmipathy | first2 = Bhaskar | last2 = Thyagarajan | name-list-style = vanc | title = Primary and Stem Cells: Gene Transfer Technologies and Applications | url = https://books.google.com/books?id=pIeqgfN1924C&pg=PA38 | isbn = 978-0-470-61074-9 | year = 2011 | edition = 1st | publisher = Wiley-Blackwell }}{{cite journal | vauthors = Arnold AS, Laporte V, Dumont S, Appert-Collin A, Erbacher P, Coupin G, Levy R, Poindron P, Gies JP | title = Comparing reagents for efficient transfection of human primary myoblasts: FuGENE 6, Effectene and ExGen 500 | journal = Fundamental & Clinical Pharmacology | volume = 20 | issue = 1 | pages = 81–9 | date = February 2006 | pmid = 16448398 | doi = 10.1111/j.1472-8206.2005.00344.x | s2cid = 42585711 }}
• Dendrimer is a class of highly branched molecules based on various building blocks and synthesized through a convergent or a divergent method. These dendrimers bind the nucleic acids to form dendriplexes that then penetrate the cells.{{Cite journal|last1=Sapra|first1=Rachit|last2=Verma|first2=Ram P.|last3=Maurya|first3=Govind P.|last4=Dhawan|first4=Sameer|last5=Babu|first5=Jisha|last6=Haridas|first6=V.|date=2019-11-13|title=Designer Peptide and Protein Dendrimers: A Cross-Sectional Analysis|journal=Chemical Reviews|volume=119|issue=21|pages=11391–11441|doi=10.1021/acs.chemrev.9b00153|pmid=31556597|s2cid=203435702|issn=0009-2665}}{{Cite journal|last1=Heitz|first1=Marc|last2=Javor|first2=Sacha|last3=Darbre|first3=Tamis|last4=Reymond|first4=Jean-Louis|date=2019-08-21|title=Stereoselective pH Responsive Peptide Dendrimers for siRNA Transfection|journal=Bioconjugate Chemistry|volume=30|issue=8|pages=2165–2182|doi=10.1021/acs.bioconjchem.9b00403|pmid=31398014|s2cid=199519310|issn=1043-1802}}

DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called 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. Lentiviral vectors are also helpful due to their ability to transduce cells not currently undergoing mitosis.

Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, electroporation) are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well.

Stable and transient transfection differ in their long term effects on a cell; a stably transfected cell will continuously express transfected DNA and pass it on to daughter cells, while a transiently transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells.

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.{{cite journal | vauthors = Kim TK, Eberwine JH | title = Mammalian cell transfection: the present and the future | journal = Analytical and Bioanalytical Chemistry | volume = 397 | issue = 8 | pages = 3173–8 | date = August 2010 | pmid = 20549496 | doi = 10.1007/s00216-010-3821-6 | pmc=2911531}} 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.{{cite journal | vauthors = Durocher Y, Perret S, Kamen A | title = High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells | journal = Nucleic Acids Research | volume = 30 | issue = 2 | pages = 9e–9 | date = January 2002 | pmid = 11788735 | pmc = 99848 | doi = 10.1093/nar/30.2.e9 }}

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.{{cite web|title=The Science of Stable Cell Line Generation|first1=Alex|last1=Fanelli | name-list-style = vanc | url = http://www.stablecellline.com/the-science-of-stable-cell-line-generation/|date=2016|access-date=23 December 2017}}

Common agents for selecting stable transfection are:

• Geneticin, or G418, neutralized by the product of the neomycin resistance gene
• Puromycin
• Zeocin
• Hygromycin B
• Blasticidin S

RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. RNA transfection is often used in primary cells that do not divide.

siRNAs can also be transfected to achieve RNA silencing (i.e. loss of RNA and protein from the targeted gene). This has become a major application in research to achieve "knock-down" of proteins of interests (e.g. Endothelin-1{{cite journal | vauthors = Mawji IA, Marsden PA | title = RNA transfection is a versatile tool to investigate endothelin-1 posttranscriptional regulation | journal = Experimental Biology and Medicine | volume = 231 | issue = 6 | pages = 704–708 | date = June 2006 | pmid = 16740984 | doi = 10.3181/00379727-231-2310704 | url = https://journals.sagepub.com/doi/abs/10.3181/00379727-231-2310704 | doi-broken-date = 1 November 2024 }}) with potential applications in gene therapy. Limitation of the silencing approach are the toxicity of the transfection for cells and potential "off-target" effects on the expression of other genes/proteins.

RNA can be purified from cells after lysis or synthesized from free nucleotides either chemically, or enzymatically using an RNA polymerase to transcribe a DNA template. As with DNA, RNA can be delivered to cells by a variety of means including microinjection, electroporation, and lipid-mediated transfection. If the RNA encodes a protein, transfected cells may translate the RNA into the encoded protein.{{cite journal | vauthors = Herb M, Farid A, Gluschko A, Krönke M, Schramm M | title = Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA | journal = Journal of Visualized Experiments | date = November 2019 | issue = 153 | doi=10.3791/60143 | pmid = 31762462 | doi-access = free }} If the RNA is a regulatory RNA (such as a miRNA), the RNA may cause other changes in the cell (such as RNAi-mediated knockdown).

Encapsulating the RNA molecule in lipid nanoparticles was a breakthrough for producing viable RNA vaccines, solving a number of key technical barriers in delivering the RNA molecule into the human cell.{{cite web | website=Stat | date=1 December 2020 | accessdate=3 December 2020 | url=https://www.statnews.com/2020/12/01/how-nanotechnology-helps-mrna-covid19-vaccines-work/ | title=How nanotechnology helps mRNA Covid-19 vaccines work | first=Elizabeth | last=Cooney}}{{cite news |last1=Foley |first1=Katherine Ellen |title=The first Covid-19 vaccines have changed biotech forever |url=https://qz.com/1948132/the-first-covid-19-vaccines-have-changed-biotech-forever/ |access-date=11 January 2021 |work=Quartz |publisher=Quartz Media |date=22 December 2020}}

RNA molecules shorter than about 25nt (nucleotides) largely evade detection by the innate immune system, which is triggered by longer RNA molecules. Most cells of the body express proteins of the innate immune system, and upon exposure to exogenous long RNA molecules, these proteins initiate signaling cascades that result in inflammation. This inflammation hypersensitizes the exposed cell and nearby cells to subsequent exposure. As a result, while a cell can be repeatedly transfected with short RNA with few non-specific effects, repeatedly transfecting cells with even a small amount of long RNA can cause cell death unless measures are taken to suppress or evade the innate immune system (see "Long-RNA transfection" below).

Short-RNA transfection is routinely used in biological research to knock down the expression of a protein of interest (using siRNA) or to express or block the activity of a miRNA (using short RNA that acts independently of the cell's RNAi machinery, and therefore is not referred to as siRNA). While DNA-based vectors (viruses, plasmids) that encode a short RNA molecule can also be used, short-RNA transfection does not risk modification of the cell's DNA, a characteristic that has led to the development of short RNA as a new class of macromolecular drugs.{{cite news | url=http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2006/08/11/BUGPNKFU6T1.DTL | publisher=San Francisco Chronicle | author=Tansey B | title=Macular degeneration treatment interferes with RNA messages | date=11 August 2006}}

Long-RNA transfection is the process of deliberately introducing RNA molecules longer than about 25nt into living cells. A distinction is made between short- and long-RNA transfection because exogenous long RNA molecules elicit an innate immune response in cells that can cause a variety of nonspecific effects including translation block, cell-cycle arrest, and apoptosis.

The innate immune system has evolved to protect against infection by detecting pathogen-associated molecular patterns (PAMPs), and triggering a complex set of responses collectively known as inflammation. Many cells express specific pattern recognition receptors (PRRs) for exogenous RNA including toll-like receptor 3,7,8 (TLR3, TLR7, TLR8),{{cite journal | doi=10.1038/35099560 |vauthors=Alexopoulou L, Holt AC, Medzhitov R, Flavell RA | title=Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3 | journal=Nature | year=2001 | volume=413 | issue=6857 | pages=732–738 | pmid=11607032|bibcode=2001Natur.413..732A |s2cid=4346537 }}{{cite journal | doi=10.1074/jbc.M310175200 |vauthors=Kariko K, Ni H, Capodici J, Lamphier M, Weissman D | title=mRNA is an endogenous ligand for Toll-like receptor 3 | journal=J Biol Chem | year=2004 | volume=279 | issue=13 | pages=12542–12550 | pmid=14729660| doi-access=free }}{{cite journal | doi=10.1016/j.immuni.2005.06.008 |vauthors=Kariko K, Buckstein M, Ni H, Weissman D | title=Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA | journal=Immunity | year=2005 | volume=23 | issue=2 | pages=165–175 | pmid=16111635| doi-access=free }}{{cite journal | doi=10.1126/science.1093616 |vauthors=Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C | title=Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA | journal=Science | year=2004 | volume=303 | issue=5663 | pages=1529–1531 | pmid=14976261|bibcode=2004Sci...303.1529D |s2cid=33144196 | doi-access=free }} the RNA helicase RIG1 (RARRES3),{{cite journal | doi=10.1038/ni1087 |vauthors=Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, etal | title=The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses | journal=Nat Immunol | year=2004 | volume=5 | issue=7 | pages=730–737 | pmid=15208624|s2cid=34876422 }} protein kinase R (PKR, a.k.a. EIF2AK2),{{cite journal |vauthors=Das HK, Das A, Ghosh-Dastidar P, Ralston RO, Yaghmai B, etal | title=Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates | journal=J Biol Chem | year=1981 | volume=256 | issue=12 | pages=6491–6495 | doi=10.1016/S0021-9258(19)69192-1 | pmid=7240221| doi-access=free }}{{cite journal |vauthors=Levin DH, Petryshyn R, London IM | title=Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes | journal=J Biol Chem | year=1981 | volume=256 | issue=14 | pages=7638–7641 | doi=10.1016/S0021-9258(19)69008-3 | pmid=6265457| doi-access=free }} members of the oligoadenylate synthetase family of proteins (OAS1, OAS2, OAS3), and others. All of these proteins can specifically bind to exogenous RNA molecules and trigger an immune response.

The specific chemical, structural or other characteristics of long RNA molecules that are required for recognition by PRRs remain largely unknown despite intense study. At any given time, a typical mammalian cell may contain several hundred thousand mRNA and other, regulatory long RNA molecules. How cells distinguish exogenous long RNA from the large amount of endogenous long RNA is an important open question in cell biology. Several reports suggest that phosphorylation of the 5'-end of a long RNA molecule can influence its immunogenicity, and specifically that 5'-triphosphate RNA, which can be produced during viral infection, is more immunogenic than 5'-diphosphate RNA, 5'-monophosphate RNA or RNA containing no 5' phosphate.{{cite journal | doi=10.1126/science.1132505 |vauthors=Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, etal | title=5'-triphosphate RNA is the ligand for RIG-I | journal=Science | year=2006 | volume=314 | issue=5801 | pages=994–997 | pmid=17038590|bibcode=2006Sci...314..964H |s2cid=22436759 | doi-access=free }}{{cite journal | doi=10.1038/nature07106 |author1=Saito T |author2=Owen DM |author3=Jiang F |author4=Marcotrigiano J |author5=Gale M, Jr. | title=Innate immunity induced by composition-dependent RIG-I recognition of Hepatitis C virus RNA | journal=Nature | year=2008 | volume=454 | issue=7203 | pages=523–527 | pmid=18548002 | pmc=2856441|bibcode=2008Natur.454..523S }}{{cite journal | doi=10.1016/j.molcel.2007.11.028 |vauthors=Takahasi K, Yoneyama M, Nishihori T, Hirai R, Kumeta H, etal | title=Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses | journal=Mol Cell | year=2008 | volume=29 | issue=4 | pages=428–440 | pmid=18242112| doi-access=free }}{{cite journal | doi=10.1016/j.immuni.2008.07.009 |vauthors=Yoneyama M, Fujita T | title=Structural mechanism of RNA recognition by the RIG-I-like receptors | journal=Immunity | year=2008 | volume=29 | issue=2 | pages=178–181 | pmid=18701081| doi-access=free }}{{cite journal | doi=10.1073/pnas.0900971106 |vauthors=Schmidt A, Schwerd T, Hamm W, Hellmuth JC, Cui S, etal | title=5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I | journal=Proc Natl Acad Sci USA | year=2009 | volume=106 | issue=29 | pages=12067–12072 | pmid=19574455 | pmc=2705279|bibcode=2009PNAS..10612067S |doi-access=free }}{{cite journal | doi=10.1016/j.immuni.2009.05.008 |vauthors=Schlee M, Roth A, Hornung V, Hagmann CA, Wimmenauer V, etal | title=Recognition of 5'-triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative strand virus | journal=Immunity | year=2009 | volume=31 | issue=1 | pages=25–34 | pmid=19576794 | pmc=2824854}} However, in vitro-transcribed (ivT) long RNA containing a 7-methylguanosine cap (present in eukaryotic mRNA) is also highly immunogenic despite having no 5' phosphate,{{cite journal |vauthors=Angel M, Yanik MF | title=Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins| journal=PLOS ONE| year=2010| volume=5 | issue = 7| doi=10.1371/journal.pone.0011756 | pages=e11756 | pmid=20668695 | pmc=2909252| bibcode=2010PLoSO...511756A| doi-access=free}} suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule.

Eukaryotic mRNA contains chemically modified nucleotides such as N⁶-methyladenosine, 5-methylcytidine, and 2'-O-methylated nucleotides. Although only a very small number of these modified nucleotides are present in a typical mRNA molecule, they may help prevent mRNA from activating the innate immune system by disrupting secondary structure that would resemble double-stranded RNA (dsRNA),{{cite journal | vauthors = Herb M, Farid A, Gluschko A, Krönke M, Schramm M | title = Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA | journal = Journal of Visualized Experiments | date = November 2019 | issue = 153 | doi=10.3791/60143 | pmid = 31762462 | doi-access = free }} a type of RNA thought to be present in cells only during viral infection.

The immunogenicity of long RNA has been used to study both innate and adaptive immunity.

Inhibiting only three proteins, interferon-β, STAT2, and EIF2AK2 is sufficient to rescue human fibroblasts from the cell death caused by frequent transfection with long, protein-encoding RNA. Inhibiting interferon signaling disrupts the positive-feedback loop that normally hypersensitizes cells exposed to exogenous long RNA. Researchers have recently used this technique to express reprogramming proteins in primary human fibroblasts.{{cite news | url=http://web.mit.edu/newsoffice/2010/reprogramming-stem-cells-0726.html| publisher=MIT News Office | author=Trafton A | title=RNA offers a safer way to reprogram cells | date=26 July 2010}}

• Gene targeting
• Minicircle
• Protofection
• Transformation
• Transduction
• Transgene
• Vector (molecular biology)
• Viral vector

{{reflist}}

{{refbegin}}

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• {{cite journal | vauthors = Bonetta L |title=The inside scoop—evaluating gene delivery methods |journal=Nature Methods |volume=2 |issue=11 |pages=875–883 |year=2005 |doi=10.1038/nmeth1105-875 |s2cid=8078059 |doi-access=free }}

{{refend}}

{{scholia}}

• {{MeSH name|Transfection}}
• [http://transfection.ws/ Biology Research Resource — Articles and Forums about Transfection]
• [https://web.archive.org/web/20091013093039/http://www.st-andrews.ac.uk/~biophot/researchopticaltransfection.html Research in optical transfection at the University of St Andrews]
• [https://web.archive.org/web/20090907020041/http://www.drug-delivery-systems.org/ The 10th US-Japan Symposium on Drug Delivery Systems]

{{Genetic recombination}}

{{Genetic engineering}}

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