Transfection is the process of deliberately introducing naked or purifiednucleic acids intoeukaryoticcells.[1][2] It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viralDNA transfer inbacteria and non-animaleukaryotic cells, including plant cells. In animal cells, transfection is the preferred term, as the term "transformation" is also used to refer to a cell's progression to a cancerous state (carcinogenesis).Transduction is often used to describe virus-mediated gene transfer into prokaryotic cells.[2][3]
The wordtransfection is aportmanteau of the prefixtrans- and the word "infection."Genetic material (such assupercoiled plasmid DNA orsiRNA constructs), may be transfected. Transfection ofanimal cells typically involves opening transient pores or "holes" in thecell membrane to allow the uptake of material. Transfection can be carried out usingcalcium phosphate (i.e.tricalcium phosphate), byelectroporation, by cell squeezing, or by mixing acationiclipid with the material to produceliposomes that fuse with the cell membrane and deposit their cargo inside.
Transfection can result in unexpected morphologies and abnormalities in target cells.
The meaning of the term has evolved.[4] The original meaning of transfection was "infection by transformation", i.e., introduction of genetic material, DNA or RNA, from aprokaryote-infecting virus orbacteriophage 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.[citation needed]
There are various methods of introducing foreignDNA into a eukaryoticcell: 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.[5]
Physical methods includeelectroporation,microinjection,gene gun,impalefection,hydrostatic pressure, continuous infusion, and sonication. Chemicals include methods such aslipofection, which is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes).[6] Biological transfection is typically mediated byviruses, 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 includeretrovirus,lentivirus,adenovirus,adeno-associated virus, andherpes simplex virus.[citation needed]
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 iselectroporation, where short electrical pulses disrupt the cell membrane, allowing the transfected nucleic acids to enter the cell.[5] Other physical methods use different means to poke holes in the cell membrane:Sonoporation uses high-intensity ultrasound (attributed mainly to thecavitation of gas bubbles interacting with nearby cell membranes),optical transfection uses a highly focused laser to form a ~1 μm diameter hole.[7]
Several methods use tools that force the nucleic acid into the cell, namely:microinjection of nucleic acid with a fine needle;[5]biolistic particle delivery, in which nucleic acid is attached to heavy metal particles (usually gold) and propelled into the cells at high speed;[8] andmagnetofection, where nucleic acids are attached to magneticiron oxide particles and driven into the target cells by magnets.[8]
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.[9]
Chemical-based transfection can be divided into several kinds:cyclodextrin,[10] polymers,[11] liposomes, or nanoparticles[12] (with or without chemical or viral functionalization. See below).
DNA can also be introduced into cells usingviruses as a carrier. In such cases, the technique is calledtransduction, 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.
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 todaughter 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 throughmitosis or degraded.[5] Cell lines expressing theEpstein–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.[25]
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, amarker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certaintoxin. 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 toproliferate, 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.[26]
| Agent | Selectable marker |
|---|---|
| Geneticin (G418) | neomycin resistance geneNeoR |
| Puromycin | Puromycin N-acetyltransferase (PURO) |
| Zeocin | Sh Ble |
| Hygromycin B | Hygromycin resistance geneHph |
| Blasticidin S | At Bsd orBc Bsr |
RNA can also be transfected into cells to transiently express its coded protein, or to studyRNA 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[27]) 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 afterlysis or synthesized from freenucleotides either chemically, or enzymatically using anRNA polymerase totranscribe aDNA template. As with DNA, RNA can be delivered to cells by a variety of means includingmicroinjection,electroporation, andlipid-mediated transfection. If the RNA encodes aprotein, transfected cells maytranslate the RNA into the encoded protein.[28] If the RNA is a regulatory RNA (such as amiRNA), the RNA may cause other changes in the cell (such asRNAi-mediated knockdown).
Encapsulating the RNA molecule inlipid nanoparticles was a breakthrough for producing viableRNA vaccines, solving a number of key technical barriers in delivering the RNA molecule into the human cell.[29][30]
RNA molecules shorter than about 25nt (nucleotides) largely evade detection by theinnate 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 ininflammation. 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 (usingsiRNA) or to express or block the activity of amiRNA (using short RNA that acts independently of the cell'sRNAi 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 ofmacromoleculardrugs.[31]
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 aninnate immune response in cells that can cause a variety of nonspecific effects includingtranslation block,cell-cycle arrest, andapoptosis.
The innate immune system has evolved to protect againstinfection by detectingpathogen-associated molecular patterns (PAMPs), and triggering a complex set of responses collectively known asinflammation. Many cells express specificpattern recognition receptors (PRRs) for exogenous RNA includingtoll-like receptor 3,7,8 (TLR3,TLR7,TLR8),[32][33][34][35] the RNAhelicaseRIG1 (RARRES3),[36]protein kinase R (PKR, a.k.a. EIF2AK2),[37][38] 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 typicalmammalian 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 incell biology. Several reports suggest thatphosphorylation of the 5'-end of a long RNA molecule can influence itsimmunogenicity, 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.[39][40][41][42][43][44] However, in vitro-transcribed (ivT) long RNA containing a7-methylguanosine cap (present ineukaryotic mRNA) is also highly immunogenic despite having no 5' phosphate,[45] suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule.
Eukaryotic mRNA contains chemically modified nucleotides such asN6-methyladenosine,5-methylcytidine, and2'-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 disruptingsecondary structure that would resemble double-stranded RNA (dsRNA),[46][34] 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 andadaptive immunity.
Inhibiting only three proteins,interferon-β,STAT2, andEIF2AK2 is sufficient to rescue humanfibroblasts from the cell death caused by frequent transfection with long, protein-encoding RNA.[45] Inhibiting interferon signaling disrupts the positive-feedback loop that normally hypersensitizes cells exposed to exogenous long RNA. Researchers have recently used this technique to expressreprogramming proteins in primary humanfibroblasts.[47]
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