HEK 293 cells

HEK 293 cells grown for several days in standard tissue culture medium.

Human embryonic kidney cells 293, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells have been widely used in cell biology research for many years, because of their reliable growth and propensity for transfection. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy.

Origin

HEK 293 cells were generated in 1973 by transformation of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb's laboratory in Leiden, the Netherlands. The human embryonic kidney cells were obtained from a single apparently healthy fetus legally aborted under Dutch law; the identity of the mother and the reason for the abortion are no longer known.[1] The kidney cells were originally cultured by van der Eb himself; the transformation by adenovirus was performed by Frank Graham, at that time a post-doc in van der Eb's lab, and were published in 1977 after Graham left Leiden for McMaster University in Canada.[2] They are called HEK since they originated in human embryonic kidney cultures, while the number 293 came from Graham's habit of numbering his experiments; the original HEK 293 cell clone was simply the product of his 293rd experiment. It should be noted that Graham performed the transformation a total of eight times, obtaining just one clone of cells which were cultured with some difficulty for several months. After presumably adapting to tissue culture, cells from this clone finally developed into the relatively stable HEK 293 line.

Subsequent analysis has shown that the transformation was brought about by an insert consisting of ~4.5 kilobases from the left arm of the viral genome, which became incorporated into human chromosome 19.[3]

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 was very inefficient, suggesting that the cell which finally produced the HEK 293 line may have been unusual in some fashion. In fact, Graham and coworkers have 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 the original kidney culture.[4] A very comprehensive study of the genomes and transcriptomes of HEK 293 and five derivative cell lines provides a wealth of new information about these cells.[5] These authors also compared the HEK 293 transcriptome with that of human kidney, adrenal, pituitary and central nervous tissue. The HEK 293 pattern clearly most closely resembled that seen in adrenal cells, which have many neuronal properties. Given the location of the adrenal gland (adrenal means "next to the kidney"), a few adrenal cells could plausibly have been found in an embryonic kidney derived culture, and could be preferentially transformed by adenovirus. In fact adenovirus has been shown to transform neuronal lineage cells much more efficiently than typical human kidney epithelial cells.[4] An embryonic adrenal precursor cell therefore seems the most likely cell of origin 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 a very 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 normal diploid human cell. Chromosomal abnormalities include a total of three copies of the X chromosome and four copies of chromosome 17 and chromosome 22.[5][6] The presence of multiple X chromosomes and the lack of any trace of Y chromosome derived sequence suggest that the original fetus was female.

Applications

Immunofluorescent HEK 293 cells

HEK 293 cells are straightforward to grow in culture and to transfect, and so have been widely used as hosts for gene expression. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analyzing the expressed protein. The widespread use of this cell line is due to its extreme transfectability by the various techniques, including calcium phosphate method, achieving efficiencies approaching 100%.

Examples of such experiments include:

A more specific use of HEK 293 cells is in the propagation of adenoviral vectors. Viruses offer an extremely efficient means of delivering genes into cells, since this is what they have evolved to do, and are thus of great use as experimental tools. However, as pathogens, they also present a degree of danger 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, 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.[12]

An important variant of this cell line is the 293T cell line that 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 the desired gene products. Note that any similarly modified cell line can be used for this sort of work; HeLa, COS and Chinese Hamster Ovary cell are common alternatives. 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.

Native proteins of interest

Depending on various conditions, 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:

References

  1. ↑ Alex van der Eb. "USA FDA CTR For Biologics Evaluation and Research Vaccines and Related Biological Products Advisory Committee Meeting" (PDF). Lines 14–22: USFDA. p. 81. Retrieved August 11, 2012.
  2. ↑ Graham FL, Smiley J, Russell WC, Nairn R (July 1977). "Characteristics of a human cell line transformed by DNA from human adenovirus type 5". J. Gen. Virol. 36 (1): 59–74. doi:10.1099/0022-1317-36-1-59. PMID 886304.
  3. ↑ Louis N, Evelegh C, Graham FL (July 1997). "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line". Virology. 233 (2): 423–9. doi:10.1006/viro.1997.8597. PMID 9217065.
  4. 1 2 Shaw G, Morse S, Ararat M, Graham FL (June 2002). "Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells". FASEB J. 16 (8): 869–71. doi:10.1096/fj.01-0995fje. PMID 11967234.
  5. 1 2 Lin YC, Boone M, Meuris L, Lemmens I, Van Roy N, Soete A, Reumers J, Moisse M, Plaisance S, Drmanac R, Chen J, Speleman F, Lambrechts D, Van de Peer Y, Tavernier J, Callewaert N (September 2014). "Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations". Nature Commun. 5 (8): 4767. doi:10.1038/ncomms5767. PMID 25182477.
  6. ↑ "ECACC Catalogue Entry for HEK 293". hpacultures.org.uk. ECACC. Retrieved 2012-03-18. External link in |work= (help)
  7. ↑ Fredj S, Sampson KJ, Liu H, Kass RS (May 2006). "Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action". Br. J. Pharmacol. 148 (1): 16–24. doi:10.1038/sj.bjp.0706709. PMC 1617037Freely accessible. PMID 16520744.
  8. ↑ Amar L, Desclaux M, Faucon-Biguet N, Mallet J, Vogel R (2006). "Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter". Nucleic Acids Res. 34 (5): e37. doi:10.1093/nar/gkl034. PMC 1390691Freely accessible. PMID 16522642.
  9. ↑ Kanno T, Yamamoto H, Yaguchi T, et al. (June 2006). "The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site". J. Lipid Res. 47 (6): 1146–56. doi:10.1194/jlr.M500329-JLR200. PMID 16520488.
  10. ↑ Li T, Paudel HK (March 2006). "Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism". Biochemistry. 45 (10): 3125–33. doi:10.1021/bi051634r. PMID 16519507.
  11. ↑ Mustafa H, Strasser B, Rauth S, Irving RA, Wark KL (April 2006). "Identification of a functional nuclear export signal in the green fluorescent protein asFP499". Biochem. Biophys. Res. Commun. 342 (4): 1178–82. doi:10.1016/j.bbrc.2006.02.077. PMID 16516151.
  12. ↑ He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B (March 1998). "A simplified system for generating recombinant adenoviruses". Proc. Natl. Acad. Sci. U.S.A. 95 (5): 2509–14. doi:10.1073/pnas.95.5.2509. PMC 19394Freely accessible. PMID 9482916.
  13. ↑ Dautzenberg FM, Higelin J, Teichert U (February 2000). "Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells". Eur. J. Pharmacol. 390 (1-2): 51–9. doi:10.1016/S0014-2999(99)00915-2. PMID 10708706.
  14. ↑ Meyer zu Heringdorf D, Lass H, Kuchar I, et al. (March 2001). "Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors". Eur. J. Pharmacol. 414 (2-3): 145–54. doi:10.1016/S0014-2999(01)00789-0. PMID 11239914.
  15. ↑ Luo J, Busillo JM, Benovic JL (April 2008). "M3 Muscarinic Acetylcholine Receptor-Mediated Signaling is Regulated by Distinct Mechanisms". Mol. Pharmacol. 74 (2): 338–47. doi:10.1124/mol.107.044750. PMID 18388243.
  16. ↑ Zagranichnaya TK, Wu X, Villereal ML (August 2005). "Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells". J. Biol. Chem. 280 (33): 29559–69. doi:10.1074/jbc.M505842200. PMID 15972814.
Wikimedia Commons has media related to HEK293 cells.
This article is issued from Wikipedia - version of the 10/10/2016. The text is available under the Creative Commons Attribution/Share Alike but additional terms may apply for the media files.