Misplaced Pages

#386613

63-603: The HEK 293 cell line has been widely used in research for decades due to its reliable and fast growth and propensity for transfection . The cell line is used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy as well as safety testing for a vast array of chemicals. HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb 's laboratory in Leiden,

126-561: 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. The meaning of the term has evolved. 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

189-483: A neo -resistance gene as a selectable marker . Currently, research is being performed to understand if derivatives of neomycin have the same antibiotic effects while still being effective against neomycin-resistant bacteria. Neomycin was first isolated from the Streptomyces fradiae and Streptomyces albogriseus in 1949 (NBRC 12773). Neomycin is a mixture of neomycin B (framycetin); and its epimer neomycin C,

252-400: 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

315-447: A cell line that expresses the missing genes is required. Since HEK 293 cells express a number of adenoviral genes, they can be used to propagate adenoviral vectors in which these genes (typically, E1 and E3) are deleted, such as AdEasy. However, homologous recombination between the inserted cellular Ad5 sequence and the vector sequence, although rare, can restore the replication capacity to the vector. An important variant of this cell line

378-469: 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

441-418: A complex karyotype , exhibiting two or more copies of each chromosome and with a modal chromosome number of 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a haploid human gamete. Chromosomal abnormalities include a total of three copies of the X chromosomes and four copies of chromosome 17 and chromosome 22 . The presence of multiple X chromosomes and

504-651: 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

567-501: A few minor compounds found in much lower quantities. Neomycin B is the most active component in neomycin followed by neomycin C and neomycin A. Neomycin A is an inactive degradation product of the C and B isomers. The quantities of these components in neomycin vary from lot-to-lot depending on the manufacturer and manufacturing process. Aminoglycosides such as neomycin are known for their ability to bind to duplex RNA with high affinity. The association constant for neomycin with A-site RNA

630-459: A four-ring structure. Three of the rings are six-membered, and one is five-membered. Neomycin B and Neomycin C are stereoisomers of each other and differ by only one stereocenter one giving the R conformation and the other giving the S conformation. Neomycin C can undergo enzymatic synthesis from ribostamycin. Standard grade neomycin is composed of several related compounds including neomycin A (neamine), neomycin B (framycetin), neomycin C, and

693-429: A high binding affinity for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid component of cell membranes. Neomycin resistance is conferred by either one of two kanamycin kinase genes. Genes conferring neomycin-resistance are commonly included in DNA plasmids used to establish stable mammalian cell lines expressing cloned proteins in culture. Many commercially available protein expression plasmids contain

SECTION 10

#1732801040387

756-466: A putative alcohol dehydrogenase gene (btrE, neo5); and another putative dehydrogenase (similar to chorine dehydrogenase and related flavoproteins) gene (btrQ, neo11). A deacetylase acting to remove the acetyl group on N-acetylglucosamine moieties of aminoglycoside intermediates (Neo16) remains to be clarified (sequence similar to BtrD). Next is the attachment of the D -ribose via ribosylation of neamine, using 5-phosphoribosyl-1-diphosphate (PRPP) as

819-428: 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

882-417: 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. There are various methods of introducing foreign DNA into

945-458: 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,

1008-481: 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), 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

1071-430: Is a portmanteau of trans- and 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

1134-517: Is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes). 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 . Physical methods are

1197-490: 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

1260-514: Is an aminoglycoside antibiotic that displays bactericidal activity against Gram-negative aerobic bacilli and some anaerobic bacilli where resistance has not yet arisen. It is generally not effective against Gram-positive bacilli and anaerobic Gram-negative bacilli. Neomycin comes in oral and topical formulations, including creams, ointments, and eyedrops. Neomycin belongs to the aminoglycoside class of antibiotics that contain two or more amino sugars connected by glycosidic bonds . Neomycin

1323-454: Is caused by ear hair cell death, which occurs in response to treatment with neomycin. Patients with existing tinnitus or sensorineural hearing loss are advised to speak with a healthcare practitioner about the risks and side effects prior to taking this medication. Neomycin's antibacterial activity stems from its binding to the 30S subunit of the prokaryotic ribosome , where it inhibits prokaryotic translation of mRNA. Neomycin also exhibits

SECTION 20

#1732801040387

1386-481: Is composed of four linked moieties: D -neosamine, 2-deoxystreptamine (2-DOS), D -ribose, and L -neosamine. Neomycin A, also called neamine, contains D -neosamine and 2-deoxystreptamine. Six genes are responsible for neamine biosynthesis: DOIS gene (btrC, neo7); L-glutamine:DOI aminotransferase gene (btrS, neo6); a putative glycosyltransferase gene (btrM, neo8); a putative aminotransferase (similar to glutamate-1-semialdehyde 2,1-aminomutase) gene (btrB, neo18);

1449-432: Is in the propagation of adenoviral vectors . Viruses offer an efficient means of delivering genes into cells, which they evolved to do, and are thus of great use as experimental tools. However, as pathogens , they also present a risk to the experimenter. This danger can be avoided by the use of viruses which lack key genes, and which are thus unable to replicate after entering a cell. In order to propagate such viral vectors,

1512-611: Is inactive at 40 °C. 293T is very efficiently transfected with DNA (like its parent HEK 293). Due to the expression of SV40 large T antigen, transfected plasmid DNAs that carry the SV40 origin of replication can replicate in 293T and will transiently maintain a high copy number; this can greatly increase the amount of recombinant protein or retrovirus that can be produced from the cells. The full genome sequences of three different isolates of 293T have been determined. They are quite similar to each other but show detectable divergence from

1575-610: 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 ),

1638-555: Is more immunogenic than 5'-diphosphate RNA, 5'-monophosphate RNA or RNA containing no 5' phosphate. 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, suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule. Eukaryotic mRNA contains chemically modified nucleotides such as N -methyladenosine , 5-methylcytidine , and 2'-O-methylated nucleotides. Although only

1701-443: Is not administered via injection, as it is extremely nephrotoxic (damaging to kidney function) even when compared to other aminoglycosides . The exception is when neomycin is included, in small quantities, as a preservative in some vaccines – typically 25 μg per dose. Similar to other aminoglycosides, neomycin has excellent activity against Gram-negative bacteria and is partially effective against Gram-positive bacteria . It

1764-443: 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 . Long-RNA transfection is the process of deliberately introducing RNA molecules longer than about 25nt into living cells. A distinction

1827-452: 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 ) 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

1890-457: Is relatively toxic to humans, with allergic reactions noted as a common adverse reaction (see: hypersensitivity ). Physicians sometimes recommend using antibiotic ointments without neomycin, such as Polysporin . The following represents minimum inhibitory concentration (MIC) susceptibility data for a few medically significant Gram-negative bacteria. In 2005–06, Neomycin was the fifth-most-prevalent allergen in patch test results (10.0%). It

1953-463: 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 . Neomycin Neomycin

HEK 293 cells - Misplaced Pages Continue

2016-552: Is the 293T cell line. It contains the SV40 large T-antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of desired gene products. HEK 293, and especially HEK 293T, cells are commonly used for the production of various retroviral vectors. Various retroviral packaging cell lines are also based on these cells. Depending on various conditions,

2079-514: Is then isolated and purified from the bacterium. Neomycin is typically applied as a topical preparation, such as Neosporin ( neomycin/polymyxin B/bacitracin ). The antibiotic can also be administered orally, in which case it is usually combined with other antibiotics. Neomycin is not absorbed from the gastrointestinal tract and has been used as a preventive measure for hepatic encephalopathy and hypercholesterolemia . By killing bacteria in

2142-410: 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 transformation is also used to refer to progression to a cancerous state ( carcinogenesis ) in these cells. Transduction is often used to describe virus-mediated gene transfer into prokaryotic cells. The word transfection

2205-491: Is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution. If it is desired that the transfected gene actually remain in

2268-459: The BOSC23 packaging cell line for producing retroviral particles. Multiple variants of HEK 293 have been reported. The transfection used to create 293T (involving plasmid pRSV-1609) conferred neomycin / G418 resistance and expression of the tsA1609 allele of SV40 large T antigen; this allele is fully active at 33 °C (its permissive temperature ), has substantial function at 37 °C, and

2331-418: The cavitation of gas bubbles interacting with nearby cell membranes), optical transfection uses a highly focused laser to form a ~1 μm diameter hole. 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

2394-550: The gene expression of HEK 293 cells may vary. The following proteins of interest (among many others) are commonly found in untreated HEK 293 cells: Alvin Wong, a Catholic bioethicist, argues that despite the uncertainty over the origin of the embryonic cells used to obtain the cell line, one can infer that it came from a voluntary abortion . To some, this may present an ethical dilemma for using HEK 293 and derivative products, such as vaccines and many medications. On 21 December 2020,

2457-502: 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

2520-400: The transformation was brought about by inserting ~4.5 kilobases from the left arm of the adenoviral genome, which became incorporated into human chromosome 19 . For many years it was assumed that HEK 293 cells were generated by transformation of either a fibroblastic , endothelial or epithelial cell , all of which are abundant in kidneys. However, the original adenovirus transformation

2583-574: The COVID-19 pandemic, anti-vaccination activists noted that HEK 293 cells are used in the manufacturing of the Oxford–AstraZeneca COVID-19 vaccine (AKA AZD1222). The cells are filtered out of the final products. Regeneron Pharmaceuticals , the maker of REGN-COV2, a therapeutic antibody cocktail used to alleviate symptoms of patients with COVID-19, did not use HEK 293T cells to produce the antibody cocktail but did use those cells to assess

HEK 293 cells - Misplaced Pages Continue

2646-492: The Netherlands . The cells were obtained from a single, aborted or miscarried fetus, the precise origin of which is unclear. The cells were cultured by van der Eb; the transfection with adenoviral DNA was performed by Frank Graham , a post-doc in van der Eb's lab. They were published in 1977 after Graham left Leiden for McMaster University . They are called HEK since they originated in human embryonic kidney cultures, while

2709-521: The RNA helicase RIG1 (RARRES3) , protein kinase R (PKR, a.k.a. EIF2AK2), 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,

2772-460: The RNA into the encoded protein. 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. RNA molecules shorter than about 25nt (nucleotides) largely evade detection by

2835-703: The Roman Catholic Congregation for the Doctrine of the Faith stated that the moral duty to avoid vaccines made from cell lines derived from fetuses is, "not obligatory if there is grave danger, such as the otherwise uncontainable spread of a serious pathological event -- in this case, the pandemic spread of SARS-CoV-2 virus that causes Covid-19". The statement then justifies the use of other vaccines, "all vaccinations recognized as clinically safe and effective can be used in good conscience..." During

2898-669: The cells at high speed; 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. Chemical-based transfection can be divided into several kinds: cyclodextrin , polymers, liposomes, or nanoparticles (with or without chemical or viral functionalization. See below). DNA can also be introduced into cells using viruses as

2961-440: 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

3024-469: The expressed protein . The widespread use of this cell line is due to its transfectability by the various techniques, including calcium phosphate method, achieving efficiencies approaching 100%. Examples of such experiments include: HEK 293 cells were adapted to grow in suspension culture, as opposed to proliferation on plastic plates, in 1985. This enabled the growth of large amounts of recombinant adenovirus vectors. A more specific use of HEK 293 cells

3087-435: 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

3150-426: 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

3213-420: The intestinal tract, Neomycin keeps ammonia levels low and prevents hepatic encephalopathy, especially before gastrointestinal surgery . Waksman and Lechevalier originally noted that neomycin was active against streptomycin-resistant bacteria as well as Mycobacterium tuberculosis , the causative agent for tuberculosis . Neomycin has also been used to treat small intestinal bacterial overgrowth . Neomycin

SECTION 50

#1732801040387

3276-540: The kidney"), a few adrenal cells could plausibly have appeared in an embryonic kidney derived culture, and could be preferentially transformed by adenovirus. Adenoviruses transform neuronal lineage cells much more efficiently than typical human kidney epithelial cells. An embryonic adrenal precursor cell therefore seems the most likely origin cell of the HEK 293 line. As a consequence, HEK 293 cells should not be used as an in vitro model of typical kidney cells. HEK 293 cells have

3339-489: The lack of any trace of Y chromosome derived sequence suggest that the source fetus was female. The 293T cell line was created in Michele Calos's lab at Stanford by stable transfection of the HEK 293 cell line with a plasmid encoding a temperature-sensitive mutant of the SV40 large T antigen ; it was originally referred to as 293/ tsA1609neo . The first reference to the cell line as "293T" may be its use to create

3402-407: The latter component accounting for some 5–15% of the mixture. It is a basic compound that is most active with an alkaline reaction. It is also thermostable and soluble in water (while insoluble in organic solvents). Neomycin has good activity against Gram-positive and Gram-negative bacteria , but is ototoxic . Its use is thus restricted to the oral treatment of intestinal infections. Neomycin B

3465-447: 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. Common agents for selecting stable transfection are: RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. RNA transfection

3528-418: The number 293 came from Graham's habit of numbering his experiments; the original HEK 293 cell clone was from his 293rd experiment. Graham performed the transfection a total of eight times, obtaining just one clone of cells that were cultured for several months. After presumably adapting to tissue culture, cells from this clone developed into the relatively stable HEK 293 line. Subsequent analysis has shown that

3591-410: The original kidney culture. A comprehensive study of the genomes and transcriptomes of HEK 293 and five derivative cell lines compared the HEK 293 transcriptome with that of human kidney, adrenal, pituitary and central nervous tissue. The HEK 293 pattern most closely resembled that of adrenal cells, which have many neuronal properties. Given the location of the adrenal gland ( adrenal means "next to

3654-457: The parental HEK 293 cell line. This mutant strain does not express of the equilibrative nucleoside transporter ENT1. The gene was knocked out using CRISPR-CAS9 and the cell line retains ENT2 expression. HEK 293 cells are straightforward to grow in culture and to transfect. They have been used as hosts for gene expression . Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analyzing

3717-426: The potency of the drug. In response to ethical concerns of vaccine production, several strategies for clinicians to discuss with their patients have been suggested. Transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells . It may also refer to other methods and cell types, although other terms are often preferred: " transformation "

3780-425: The ribosyl donor (BtrL, BtrP); glycosyltransferase (potential homologues RibF, LivF, Parf) gene (Neo15). Neosamine B ( L -neosamine B) is most likely biosynthesized in the same manner as the neosamine C ( D -niosamine) in neamine biosynthesis, but with an additional epimerization step required to account for the presence of the epimeric neosamine B in neomycin B. Neomycin B and C are 23-carbon molecules with

3843-553: Was discovered in 1949 by microbiologist Selman Waksman and his student Hubert Lechevalier at Rutgers University . Neomycin received approval for medical use in 1952. Rutgers University was granted the patent for neomycin in 1957. Neomycin was discovered in 1949 by the microbiologist Selman Waksman and his student Hubert Lechevalier at Rutgers University . It is produced naturally by the bacterium Streptomyces fradiae . Synthesis requires specific nutrient conditions in either stationary or submerged aerobic conditions. The compound

SECTION 60

#1732801040387

3906-404: Was inefficient, suggesting that the cell that finally produced the HEK 293 line may have been unusual in some fashion. Graham and coworkers provided evidence that HEK 293 cells and other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons , suggesting that the adenovirus preferentially transformed a neuronal lineage cell in

3969-452: Was named Allergen of the Year in 2010. Neomycin is also a known GABA gamma -Aminobutyric acid antagonist and can be responsible for seizures and psychosis. Like other aminoglycosides, neomycin has been shown to be ototoxic , causing tinnitus , hearing loss, and vestibular problems in a small number of patients. Neomycin affects the cochlea, which is found in the inner ear. Hearing loss

#386613