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Transfection
Transfection
Transfection
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Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.[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-viral DNA transfer in bacteria and non-animal eukaryotic 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 word transfection is a portmanteau of the prefix trans- and the word "infection." Genetic material (such as supercoiled plasmid DNA or siRNA constructs), 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 that fuse with the cell membrane and deposit their cargo inside.

Transfection can result in unexpected morphologies and abnormalities in target cells.

Terminology

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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 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.[citation needed]

Methods

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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.[5]

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).[6] 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.[citation needed]

Physical methods

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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.

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.[5] 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.[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] and magnetofection, where nucleic acids are attached to magnetic iron 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 methods

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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).

  • One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[13] (see also[14]). 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.[15]
  • 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.
  • 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.[16] Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material.[17] 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.[18]
  • 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.[19][20][21][22]
  • 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.[23][24]

Viral methods

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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.[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

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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.[5] 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.[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, 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.[26]

Common agents for selecting stable transfection
Agent Selectable marker
Geneticin (G418) neomycin resistance gene NeoR
Puromycin Puromycin N-acetyltransferase (PURO)
Zeocin Sh Ble
Hygromycin B Hygromycin resistance gene Hph
Blasticidin S At Bsd or Bc Bsr

RNA transfection

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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[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 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.[28] 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.[29][30]

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.[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 an innate immune response in cells that can cause a variety of nonspecific effects including translation block, cell-cycle arrest, and apoptosis.

Endogenous vs. exogenous long RNA

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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),[32][33][34][35] the RNA helicase RIG1 (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 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.[39][40][41][42][43][44] 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,[45] 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),[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 and adaptive immunity.

Repeated long-RNA transfection

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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.[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 express reprogramming proteins in primary human fibroblasts.[47]

See also

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References

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Further reading

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Transfection

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from Grokipedia
Transfection is the deliberate introduction of foreign nucleic acids, such as DNA or RNA, into eukaryotic cells to modify their genetic content, enable gene expression, or study cellular functions. This technique refers to non-viral methods, distinct from viral transduction which uses infectious viral vectors like adenoviruses or lentiviruses to deliver genetic material. Developed as a cornerstone of molecular biology since the mid-20th century, transfection allows researchers to transiently or stably alter cell behavior, facilitating applications from basic gene function analysis to therapeutic interventions. Transfection methods are broadly classified into physical and chemical categories, each with varying efficiency, cytotoxicity, and suitability for different cell types. Physical approaches, such as —which uses electric pulses to create temporary pores in cell membranes—and , offer precise delivery but can damage cells due to mechanical stress. Chemical methods, including lipofection with cationic lipids like and calcium phosphate precipitation, form complexes with nucleic acids to facilitate uptake via , providing a balance of ease and moderate efficiency for adherent or suspension cells. In research and , transfection underpins key advancements, including the production of recombinant proteins in cell lines like HEK293 and for biopharmaceuticals, RNA for with siRNA, and delivery of CRISPR-Cas9 components for . Transient transfection yields short-term expression ideal for rapid functional assays, while stable transfection integrates DNA into the host for long-term studies or cell line engineering, often selected via antibiotic resistance markers. Despite challenges like low efficiency in primary cells and potential off-target effects, ongoing innovations in carriers and devices continue to enhance transfection's precision and applicability in for diseases such as cancer and genetic disorders.

Terminology and Fundamentals

Definition and Principles

Transfection is the process of introducing exogenous nucleic acids, such as DNA or RNA, into eukaryotic cells using non-viral methods to enable gene expression, functional studies, or genetic modification. This technique differs from viral transduction, which relies on viral vectors for delivery and often integrates genetic material into the host genome, potentially eliciting immune responses. In contrast to bacterial transformation, where prokaryotic cells uptake naked DNA for heritable changes, transfection targets eukaryotic cells and typically does not result in permanent genomic integration unless specifically designed for stable outcomes. The primary goal is to overcome cellular barriers to allow the nucleic acids to reach their functional sites, facilitating applications in research and therapy. The underlying principles of transfection revolve around cellular uptake, intracellular trafficking, and processing. Eukaryotic cells present formidable barriers, including the plasma membrane's , which repels hydrophilic s. For chemical methods, delivery aids form complexes with s to promote entry primarily via . Once internalized via , the s are often trapped in endosomes, where endosomal escape is crucial to prevent degradation by lysosomal enzymes and enable progression to the or nucleus. For DNA, successful expression requires nuclear entry, which can occur through nuclear pore complexes or during when the disassembles, influenced by DNA size and nuclear localization signals. , such as mRNA, bypasses nuclear entry and is directly translated by ribosomes in the , allowing faster onset of protein production. Efficiency of transfection is modulated by several factors, including —where adherent cell lines like HEK293 exhibit higher rates than primary or non-dividing cells—and characteristics, such as size, with larger constructs (>10 kb) reducing uptake and expression due to steric hindrance. Key metrics include transfection rate, defined as the percentage of cells successfully incorporating and expressing the , often quantified via , and expression level, measured using reporter genes like (GFP) to visualize fluorescent cells under or assess intensity proportional to protein output. For instance, uses electric pulses to transiently permeabilize the , allowing direct uptake into the and bypassing endocytic pathways. Outcomes can be transient, with episomal expression fading over divisions, or stable through selection for integration.

Historical Development

The concept of introducing exogenous DNA into mammalian cells emerged in the early , with Elisabeth Szybalska and Wacław Szybalski demonstrating the first DNA-mediated heritable transformation of a biochemical trait in human cell lines deficient in (HGPRT), using DNA extracted from wild-type cells to restore enzymatic function. This pioneering work laid the groundwork for non-viral gene transfer, though efficiencies were low and limited to specific selectable markers. Concurrently, bacterial systems advanced with Mandel and Higa's 1970 discovery of treatment to enhance competence for DNA uptake in , enabling efficient transformation that became a cornerstone for experiments. The 1970s marked a pivotal shift with the advent of recombinant DNA technology, pioneered by Stanley Cohen and Herbert Boyer's 1973 construction of biologically functional bacterial plasmids in vitro, which relied on transformation techniques to propagate hybrid DNA molecules and fueled the biotechnology revolution. In mammalian cells, Frank Graham and Arie van der Eb introduced the calcium phosphate precipitation method in 1973, allowing assay of adenovirus DNA infectivity by facilitating DNA uptake into human and monkey cell lines with improved efficiency over prior approaches, though still yielding only 1-2% transfection rates. Diethylaminoethyl (DEAE)-dextran, initially described by McCutchan and Pagano in 1968 for enhancing simian virus 40 DNA infectivity, gained widespread adoption in the 1980s for transient transfections due to its simplicity in promoting endocytosis of DNA complexes. The 1980s and 1990s saw diversification of physical methods, with Eberhard Neumann and colleagues reporting in 1982 the use of high-voltage electric pulses () to permeabilize mouse lymphoma cell membranes for DNA uptake, achieving up to 10-fold higher transformation than chemical methods and spurring refinements in pulse parameters for broader cell types by the 1990s. Chemical innovations included Philip Felgner's 1987 development of lipofection using synthetic cationic like DOTMA to form liposome-DNA complexes, enabling high-efficiency (up to 50%) transfection in diverse mammalian lines with reduced toxicity compared to precipitates. The 2000s expanded to RNA delivery following and Craig Mello's 1998 demonstration of (RNAi) via double-stranded RNA in C. elegans, which earned the 2006 and prompted protocols for synthetic siRNA transfection using or to silence genes transiently. Transfection evolved from low-efficiency, labor-intensive techniques to high-throughput methods compatible with and large-scale production, driven by recombinant DNA's demand for rapid screening. Post-2010 advances integrated CRISPR-, with and Emmanuelle Charpentier's 2012 programmable endonuclease enabling precise genome editing via or lipofection of guide RNAs and Cas9 plasmids in human cells. Regulatory milestones included the FDA's 2017 approvals of Luxturna (AAV-based retinal produced via transfection) and Kymriah (CAR-T cells transduced ), validating transfection in therapeutic manufacturing and accelerating clinical translation.

Transfection Methods

Physical Methods

Physical methods of transfection rely on mechanical or electrical forces to permeabilize cell membranes and facilitate the entry of nucleic acids, bypassing the need for chemical carriers or viral vectors. These approaches are particularly valuable for transfecting difficult cell types, such as primary cells or tissues with thick extracellular matrices, where other methods may fail. Key techniques include , , biolistics, and emerging laser-based variants like optoporation. Electroporation involves applying short, high-voltage electric pulses to cells in suspension, creating transient pores in the plasma membrane through which DNA or RNA can enter. The process was first demonstrated in 1982 by Neumann and colleagues, who showed that electric fields of 8 kV/cm for 5 microseconds dramatically enhanced DNA uptake in mouse lyoma cells. Typical parameters include voltages of 100-1000 V across a cuvette gap of 0.1-0.4 cm, resulting in field strengths of 250-2000 V/cm, and pulse durations of 1-10 ms, often using exponential decay waveforms where the time constant τ is determined by τ = RC (R being the resistance of the medium and C the capacitance of the electroporator). For optimal efficiency, cells are prepared in a low-conductivity buffer to minimize heating, harvested at 70-90% confluency, and resuspended at 10^5-10^7 cells/mL; post-transfection, cells are immediately diluted in recovery medium and incubated for 24-48 hours to allow membrane resealing and expression. This method achieves transfection efficiencies of 50-90% in hard-to-transfect cells like primary neurons, but high voltages can reduce viability to below 50% due to irreversible electroporation and Joule heating. Unlike chemical methods, electroporation leaves no residues, making it suitable for downstream applications sensitive to contaminants. Microinjection delivers nucleic acids directly into the cell cytoplasm or nucleus using a fine glass micropipette under microscopic guidance, ensuring precise targeting of individual cells. Pioneered for genetic transformation in the late 1970s, a seminal application was reported in 1980 by Gordon et al., who microinjected purified DNA into mouse embryos to achieve stable integration. The technique requires immobilizing cells on a chamber, inserting a needle with an inner diameter of 0.5-1 μm filled with 1-10 μg/μL DNA, and injecting 1-10 pL per cell at pressures of 10-100 hPa to avoid bursting. Post-injection, cells are returned to culture medium for recovery, often with serum supplementation to mitigate stress. While nearly 100% efficient for single-cell transfection, it is labor-intensive, limited to low throughput (hundreds of cells per hour), and carries risks of mechanical damage leading to 20-50% cell death, though it excels for rare or precious samples like oocytes. Biolistics, or particle bombardment, accelerates DNA-coated microprojectiles into cells using a gene gun, penetrating cell walls and membranes via kinetic energy. Developed in the mid-1980s by Sanford and colleagues at Cornell University, the method was first detailed in a 1987 Nature paper by Klein et al., demonstrating delivery of nucleic acids into living plant and animal cells with particles fired at velocities of 300-600 m/s. Gold or tungsten particles (0.5-3 μm diameter) are coated with 1-5 μg DNA/mg, loaded into a cartridge, and propelled by helium pressure (200-600 psi); target cells are plated on Petri dishes, and post-bombardment, excess particles are washed away before incubation in antibiotic-free medium for 24-72 hours. Efficiencies range from 10-50% in adherent cells, with advantages in transfecting thick tissues like skin or plant leaves without dissociation, but disadvantages include variable penetration depth causing uneven delivery and potential tissue trauma from high-impact particles. Recent advancements include laser-based optoporation, which uses focused laser pulses (typically 800 nm, 100 fs duration) to induce localized membrane poration without electrodes or needles, achieving single-cell precision. Reviews from the highlight efficiencies of 50-80% with viabilities above 70% in mammalian cells, as shown in studies like Stevenson et al. (2013) using titanium-sapphire lasers for delivery. These physical methods collectively offer high specificity and broad applicability but require optimization to balance efficiency and .

Chemical Methods

Chemical methods of transfection utilize synthetic or biochemical agents to facilitate the entry of nucleic acids into cells through the formation of protective complexes that interact with cellular membranes. These approaches rely on electrostatic interactions between positively charged carriers and negatively charged nucleic acids, promoting and subsequent intracellular release. Unlike physical methods, which may induce higher cell stress, chemical strategies emphasize and ease of use in laboratory settings. One of the most established chemical techniques is lipofection, introduced by Felgner et al. in 1987, which employs cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) to form liposomes or lipid nanoparticles that encapsulate DNA or RNA. These lipids self-assemble into vesicles via hydrophobic interactions, with the positively charged head groups condensing nucleic acids at an optimal nitrogen-to-phosphate (N/P) ratio of 2-6, where N represents moles of protonatable nitrogens in the lipid and P denotes moles of phosphates in the nucleic acid. The complexes fuse with or are endocytosed by the cell membrane, releasing the cargo in the cytoplasm, though endosomal entrapment remains a challenge. Complex assembly typically involves mixing lipids and nucleic acids in serum-free media for 15-30 minutes at room temperature, followed by adding the mixture to cells for 2-4 hours before replacing with complete media to enhance efficiency. Calcium phosphate precipitation, pioneered by Graham and van der Eb in 1973, represents another foundational chemical method, where DNA is co-precipitated with calcium ions in a buffered phosphate solution at pH 6.8-7.4 to form microcrystals that are taken up by cells via endocytosis or phagocytosis. The protocol entails dissolving DNA in a calcium chloride solution, adding it dropwise to a HEPES-buffered phosphate buffer to generate the precipitate, which is then applied to cells for 4-16 hours in a controlled environment to avoid excessive precipitation that could damage cells. This technique is particularly suited for adherent cells and large-scale transfections due to its simplicity and low cost. Polyfection using polymers like branched polyethylenimine (PEI, typically 25 kDa) offers high efficiency through the formation of polyplexes that protect nucleic acids and promote endosomal escape via the proton sponge effect, where PEI's multiple amines buffer endosomal acidification, causing osmotic swelling and rupture to release the cargo. First demonstrated by Boussif et al. in 1995, PEI complexes are prepared by mixing polymer and DNA at an N/P ratio of 5-10, incubating for 15-30 minutes, and exposing cells for 2-4 hours in serum-free conditions to optimize uptake. To mitigate PEI's cytotoxicity from high charge density, biodegradable variants or lower doses are employed, balancing efficiency with cell viability. Overall, chemical methods are cost-effective and scalable for high-throughput applications, achieving transfection efficiencies of 10-90% depending on and optimization, though variability arises from endosomal barriers and potential . Advances in nanoparticles post-2020, as seen in formulations, have improved stability and delivery, reducing compared to viral alternatives; recent developments as of 2024 include ionizable nanoparticles achieving >90% efficiency in diverse cell types for therapeutic mRNA delivery.

Viral Methods

Viral methods of transfection utilize genetically modified viruses as vectors to deliver nucleic acids into host cells, exploiting the natural mechanisms of viruses for efficient gene transfer. These vectors are engineered to carry therapeutic or experimental DNA or RNA while minimizing pathogenicity, allowing for targeted expression in various cell types. Unlike non-viral approaches, viral vectors can achieve high transduction rates by leveraging viral replication machinery and , though they require careful handling. Common viral vectors include adenoviral, lentiviral, and (AAV) systems, each with distinct properties suited to different applications. Adenoviral vectors are non-integrating, remaining episomal in the host , which enables without risking permanent genetic alteration. They can achieve high titers up to 10^12 plaque-forming units per milliliter (PFU/mL) and efficiently infect both dividing and non-dividing cells due to their broad . Lentiviral vectors, derived from HIV-1, integrate the into the host via reverse transcription of their payload, providing stable, long-term expression ideal for applications requiring persistent gene activity. These vectors are often pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) envelope to confer broad , allowing infection of a wide range of cell types, including non-dividing cells like neurons. AAV vectors, which are parvoviruses requiring helper viruses for replication, persist primarily as episomes with low integration rates, supporting long-term expression—up to five years in some tissues like muscle—without eliciting strong immune responses. AAV s 1 through 9 vary in tissue , with 2 commonly used for its efficiency in dividing cells and 9 for hepatic targeting. Production of viral vectors typically involves transient transfection of packaging cells, such as HEK293 cells, which express necessary viral proteins to assemble infectious particles from the vector genome. The multiplicity of infection (MOI), defined as the ratio of viral particles to target cells (ranging from 1 to 100), is optimized to maximize yield while minimizing during propagation. Post-production, vectors are purified using cesium chloride (CsCl) density gradient to remove contaminants and achieve high purity, ensuring safety for downstream applications. Viral methods offer advantages such as near-100% transduction efficiency in permissive cells and inherent cell-type specificity through natural , making them superior for delivery compared to non-replicating non-viral systems. However, they carry risks including from genomic integration (particularly with lentiviruses), which can disrupt host genes and potentially lead to oncogenesis, and immune responses triggered by viral proteins that may limit repeat dosing. Most viral vectors, excluding those with replication-competent elements, are handled under 2 (BSL-2) conditions to mitigate risks. The foundational use of viral transduction dates to the 1970s, when retroviruses were first demonstrated to transfer cellular genes, laying the groundwork for engineered vectors. Advances in viral delivery have extended to CRISPR-Cas9 systems, with early clinical explorations in the mid-2010s enabling precise ; for instance, CRISPR-Cas9 editing entered clinical trials by 2018 for applications like treatment. Immunogenicity in adenoviral vectors is mitigated through "gutless" or helper-dependent designs, which eliminate most viral genes to reduce capsid-associated immune activation while maintaining high-capacity delivery.

Types of Transfection Outcomes

Transient Transfection

Transient transfection refers to the temporary introduction of nucleic acids, such as DNA or mRNA, into eukaryotic cells, resulting in short-term without integration into the host . This process enables the expression of transgenes from extrachromosomal elements, typically lasting from 1 to 7 days, and is widely used for rapid functional studies in . Unlike stable transfection, which involves genomic incorporation for long-term , transient transfection relies on non-integrative delivery methods that prioritize speed and ease over persistence. The ephemerality of transient transfection arises primarily from extrachromosomal expression, where delivered plasmids remain as episomes in the nucleus and are diluted during cell division, leading to a half-life of approximately 24-48 hours in dividing cells. Additionally, nucleases in the cellular environment degrade the introduced nucleic acids over time, further limiting expression duration; for instance, plasmid DNA is progressively lost without replication signals, while mRNA is subject to rapid turnover by exonucleases. Detection of transient expression often employs reporter genes like luciferase, where activity peaks between 24 and 72 hours post-transfection, allowing quantification via luminescence assays that correlate with transgene levels. Protocols for transient transfection typically involve high doses of nucleic acids, ranging from 1 to 10 μg per 10^6 cells, to achieve sufficient expression levels despite the short window, with optimal timing for analysis set around 24-48 hours to capture peak activity. These methods are particularly suited for applications, such as in 96-well plate formats, where lipofection or delivers constructs en masse for functional assays like protein interaction studies. Efficiency can be enhanced by incorporating elements like the enhancer, which promotes episomal replication in certain cell types, thereby extending expression slightly without stable integration. A key advantage of transient transfection is its rapid readout, facilitating quick functional validation in research settings, though it suffers from low persistence, with expression often undetectable after one week due to dilution and degradation. In therapeutic contexts, transient mRNA transfection has shown superiority over DNA-based approaches in , particularly in post-2018 CAR-T cell engineering, where of mRNA yields transient receptor expression that minimizes tonic signaling and enhances safety profiles in clinical trials.

Stable Transfection

Stable transfection refers to the process by which exogenous nucleic acids, typically DNA, are integrated into the host cell's genome, enabling long-term, heritable expression of the transgene across subsequent cell divisions. This contrasts with transient transfection by achieving permanent genomic incorporation, primarily through two mechanisms: homologous recombination, which allows precise insertion at targeted loci using homology arms, or random insertion, often facilitated by non-homologous end joining (NHEJ) or transposon systems. Transposon-based methods, such as the Sleeping Beauty system—a synthetic transposon reconstructed from inactive fish elements—promote efficient cut-and-paste integration, typically resulting in 1-10 copies per cell, which influences expression levels and potential genotoxicity. The first demonstrations of stable transfection in mammalian cells occurred in the early 1970s using calcium phosphate-mediated DNA uptake, leading to the establishment of transformed lines like HEK293. More recently, CRISPR-Cas9 technologies from the Zhang laboratory have advanced precise stable knock-ins via homology-directed repair (HDR), as detailed in protocols for genome engineering published in 2013. Protocols for stable transfection begin with the delivery of linear or circular plasmids containing the and a , such as the neomycin phosphotransferase gene conferring resistance to (Geneticin). Following transfection, cells are cultured in selective media containing 200-800 μg/mL for 2-4 weeks to eliminate non-integrated cells, allowing only those with stable integrations to proliferate. Clonal isolation is then achieved through limiting dilution or fluorescence-activated (FACS) to obtain monoclonal populations, followed by verification of integration via analysis, which confirms copy number and site specificity. Recent CRISPR-mediated approaches enhance specificity by co-delivering , , and donor templates, achieving knock-in efficiencies up to several percent in optimized systems, though overall stable transfection efficiency remains below 1% without enrichment strategies like FACS. The primary advantage of stable transfection is the generation of heritable cell lines for sustained and functional studies, essential for applications like recombinant therapeutics. However, it carries risks including position effect variegation, where transgene silencing occurs due to spreading at random integration sites, and potential oncogenesis from disrupting proto-oncogenes or tumor suppressors. These drawbacks necessitate careful site selection and validation to mitigate variability in expression stability.

RNA-Specific Transfection

Endogenous vs. Exogenous RNA Delivery

Endogenous RNA molecules are produced within cells through transcription by , followed by co- and post-transcriptional processing that includes the addition of a 5' cap structure (7-methylguanosine) to protect against exonucleases and facilitate initiation, as well as the attachment of a 3' poly(A) tail by poly(A) polymerase to enhance stability and export from the nucleus. These modifications contribute to mRNA half-lives ranging from hours to days, with stability further regulated by microRNAs (miRNAs) that bind to the 3' (UTR) to inhibit or promote decay via the . In contrast, exogenous RNA for transfection is typically synthesized via in vitro transcription (IVT) using bacteriophage polymerases like T7, which lacks the cellular processing machinery, resulting in uncapped, non-polyadenylated transcripts prone to rapid degradation by ubiquitous RNases in the extracellular and intracellular environments. To mitigate innate immune activation and enhance stability, synthetic RNAs incorporate modified nucleosides such as , which reduces recognition by Toll-like receptor 3 (TLR3) and other sensors by altering RNA secondary structure and evading endolysosomal processing. RNA transfection was pioneered in 1989 by et al., who demonstrated efficient delivery using cationic liposomes, though exogenous RNAs often trigger immune responses via the RIG-I pathway, leading to production upon detection of 5' triphosphate ends. Design strategies for exogenous RNA address these challenges by mimicking endogenous features: anti-reverse cap analogs (ARCA), which incorporate a modified 7-methylguanosine during IVT to ensure correct orientation and improve translation efficiency by approximately twofold (up to 100%), are commonly used alongside enzymatic poly(A) tail addition. Optimization of the 3' UTR sequences, such as incorporating stabilizing elements from highly expressed cellular mRNAs, further boosts by enhancing ribosome recruitment and reducing decay rates. Delivery methods like can achieve transfection efficiencies up to 90% in certain cell types by transiently permeabilizing membranes, though nucleoside modifications like —advanced post-2015 and critical for mRNA vaccines—remain essential to suppress immunogenicity and enable high-level protein expression.

Repeated Long-RNA Transfection Protocols

Repeated long-RNA transfection protocols involve iterative delivery of synthetic (mRNA) molecules, typically longer than 1,000 nucleotides, to achieve sustained protein expression over extended periods without genomic integration. These methods address the limitations of single-dose transient transfection, where expression typically declines within 24-72 hours due to mRNA degradation and dilution during . By administering multiple doses, protocols enable cumulative protein accumulation that can mimic stable transfection outcomes, lasting weeks to months depending on the frequency and RNA stability enhancements. Common protocols employ daily or every-48-hour dosing schedules using lipid-based carriers like MessengerMAX, with typical amounts ranging from 1-5 μg per 10^6 cells to balance efficiency and . For instance, in stem cell reprogramming applications refined during the 2010s, fibroblasts are transfected daily for 12-18 days with a cocktail of modified mRNAs encoding reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc, at doses of approximately 100-400 ng per well in a 96-well plate format. This iterative approach yields high-efficiency generation, with pluripotency markers appearing after 10-14 doses. Weekly dosing variants have been explored for less frequent interventions in primary cell cultures, maintaining expression levels through optimized modifications like substitution to enhance stability. Key challenges in repeated long-RNA transfection include immune activation and cellular from cumulative carrier exposure, which can lead to reduced transfection efficiency or "immune fatigue" characterized by attenuated responses to subsequent doses due to pathway upregulation. strategies involve low-dose escalation—starting at 0.5-1 μg per 10^6 cells and increasing gradually—to minimize innate immune sensing via Toll-like receptors, alongside chemical modifications such as 5-methylcytidine to suppress production. Off-target effects, such as unintended silencing of endogenous genes from repeated uptake, are monitored through quantitative PCR (qPCR) assays that track transfected levels relative to genes like GAPDH, revealing peak accumulation after 3-5 doses before plateauing. In neural progenitor cells, for example, repetitive daily transfections reduced when spaced 24 hours apart after sufficient differentiation, allowing sustained expression without significant cell loss. These protocols offer advantages such as avoidance of insertional mutagenesis risks associated with viral methods, making them ideal for therapeutic protein replacement in regenerative medicine, where repeated mRNA delivery (e.g., every 10 days for multiple cycles) sustains deficient enzymes like factor IX in hemophilia B models, with effects observed over intervals up to 3 months. However, disadvantages include the logistical burden of multiple interventions, potential for variable bioavailability in vivo, and higher costs compared to single-dose alternatives. Recent preclinical studies have explored repeated mRNA delivery for cardiac regeneration, demonstrating improved tissue function in animal models without genomic integration concerns.

Applications and Advances

Research Applications

Transfection serves as a fundamental tool in gene function studies, enabling researchers to investigate protein roles through overexpression or knockdown techniques. For instance, transient overexpression of via plasmid transfection allows assessment of gain-of-function effects, while (siRNA) delivery facilitates targeted to elucidate loss-of-function phenotypes. These methods have been pivotal in dissecting cellular processes, such as the use of siRNA transfection to knockdown overexpressed genes in tumor progression models. In pathway mapping, transfection is employed to activate or inhibit specific signaling cascades, providing insights into regulatory networks. A representative example is the transient transfection of pathway components or reporters, which enables real-time monitoring of activation dynamics in response to stimuli, revealing interactions like those suppressed by Clara cell 10-kDa protein gene transfer. further leverages transfection for large-scale ; CRISPR libraries delivered via transfection into up to 10^6 cells per screen identify genetic modifiers of phenotypes, as demonstrated in imaging-based pooled assays that quantify multiplexed gene edits. Common model systems include adherent cell lines like HEK293, which achieve transfection efficiencies of approximately 70% with lipid-based reagents, making them ideal for rapid prototyping of gene effects. For more physiologically relevant contexts, 3D s are transfected using microfluidic encapsulation in microbeads, allowing clonal expansion and transgene expression with efficiencies exceeding 50% in organoid models. In animal models, transfects embryos, such as mouse neural progenitors, to study developmental gene functions with up to 80% targeting precision. Recent innovations highlight transfection's evolution in research precision. , advanced in the 2010s, relies on viral or non-viral transfection to express light-sensitive opsins in neurons, enabling millisecond control of neural circuits as reviewed in foundational studies. Single-cell transfection via nanopipette , developed around 2020, delivers precise cargos like components to individual cells with >90% viability, facilitating high-resolution functional assays. The 2006 for underscored transfection's role in siRNA delivery, transforming knockdown studies from model organisms to mammalian systems. Throughput has advanced with automated 384-well platforms, enabling parallel transfections of thousands of conditions for screens.

Therapeutic Applications

Transfection plays a pivotal role in therapeutic applications, particularly in , where nucleic acids are delivered to correct genetic defects or modulate cellular functions in patients. The first approved utilizing transfection was Glybera (alipogene tiparvovec), an (AAV)-based treatment for , authorized by the in 2012 but withdrawn in 2017 due to commercial reasons. This marked a milestone in clinical translation, demonstrating the feasibility of viral vector-mediated gene delivery for rare metabolic disorders. Subsequent approvals, such as Zolgensma () in 2019 by the U.S. Food and Drug Administration for (SMA) in children under two years, further advanced AAV-based therapies by delivering functional gene copies to motor neurons, achieving sustained motor function improvements in clinical trials. In 2023, the FDA approved Casgevy (exagamglogene autotemcel), the first CRISPR-based therapy for and transfusion-dependent beta-thalassemia, using —a non-viral physical method—to deliver CRISPR-Cas9 components to patient-derived hematopoietic stem cells , enabling high-efficiency editing with reduced risks. In vaccine development and , transfection enables rapid and targeted delivery. The Pfizer-BioNTech (BNT162b2), authorized in late 2020, relies on lipid nanoparticles to transfect mRNA encoding the into host cells, eliciting robust immune responses with 95% efficacy against symptomatic infection in phase 3 trials. Similarly, chimeric antigen receptor (CAR) T-cell engineering often incorporates transient transfection for enhanced safety, avoiding permanent genomic integration risks associated with viral methods; for instance, mRNA nanocarriers have been used to transiently express CARs in circulating T cells, reducing off-target effects and in preclinical models of solid tumors. Despite these successes, therapeutic transfection faces significant challenges, including efficient delivery and safety concerns. Hydrodynamic injection, a non-viral method for liver-targeted delivery, achieves 10-40% hepatocyte transfection efficiency in models but is limited by procedural invasiveness and lower in humans. Off-target effects, such as unintended genomic alterations from CRISPR-Cas9 editing, and the need for good manufacturing practice (GMP) production to ensure and purity remain hurdles, as evidenced by regulatory requirements for AAV therapies. Recent advances highlight non-viral nanoparticles for delivery in clinical trials, with lipid-based systems entering phase 1/2 studies in the 2020s for conditions like hereditary transthyretin-mediated , offering improved safety over viral vectors by minimizing . Post-2020 mRNA therapeutics have expanded to cancer vaccines, where personalized neoantigen-encoding mRNAs combined with checkpoint inhibitors have shown promising tumor regression in phase 1 trials for and . Ethical considerations distinguish therapies, which affect only the patient and are widely accepted, from germline editing, which risks heritable changes and is prohibited in clinical settings due to consent and equity issues for future generations.

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