Biology:HEK 293 cells

From HandWiki
Short description: Cell line derived from human embryonic kidney cells

Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific immortalised cell line derived from a spontaneously miscarried or aborted fetus or human embryonic kidney cells grown in tissue culture taken from a female fetus in 1973.[1][2]

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 as well as safety testing for a vast array of chemicals.

293T (or HEK 293T) is a derivative human cell line that expresses a mutant version of the SV40 large T antigen. It is very commonly used in biological research for making proteins and producing recombinant retroviruses.

History

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, the Netherlands. The cells were obtained from a single, aborted or miscarried fetus, the precise origin of which is unclear.[3][2] The cells were cultured by van der Eb; the transduction by adenovirus 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.[4] 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 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 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.[5]

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 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 the original kidney culture.[6]

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.[7] 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 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.[6] 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 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.[7][8] The presence of multiple X chromosomes and 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.[9] The first reference to the cell line as "293T" may be its use to create the BOSC23 packaging cell line for producing retroviral particles.[10]

Variants

Multiple variants of HEK 293 have been reported.


HEK 293T

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 is inactive at 40 °C.[11] 293T is very efficiently transfectable 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 the parental HEK 293 cell line.[12]

Applications

Immunofluorescent HEK 293 cells

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 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.[18] This enabled the growth of large amounts of recombinant adenovirus vectors.

A more specific use of HEK 293 cells is in the propagation of adenoviral vectors.[19] 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, 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.[20] However, homologous recombination between the inserted cellular Ad5 sequence and the vector sequence, although rare, can restore the replication capacity to the vector.[21]

An important variant of this cell line 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.[22] 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:

Bioethics

Alvin Wong argues that despite the uncertainty over the origin of the fetus used to obtain the cell line, circumstantial evidence strongly suggests that it came from a voluntary abortion. To some Catholics and Eastern Orthodox Christians, this presents an ethical dilemma for using HEK 293 and derivative products, such as vaccines and many medications.[27][28][29][30]

On 21 December 2020, the Roman Catholic Congregation for the Doctrine of the Faith, with the approval of the Pope, stated that "it is morally acceptable to receive COVID-19 vaccines that have used cell lines from aborted fetuses in their research and production process", but only where other alternatives are non-existent (or currently unavailable), or when there is the risk of a worse danger.[31]

During 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.[32]

According to pharmaceutical companies, such as Regeneron Pharmaceuticals, the modern cell line is so far removed from its origin that it is no longer considered fetal tissue.[33][28]

See also

References

  1. ↑ Kavsan, Vadym M; Iershov, Anton V; Balynska, Olena V (23 May 2011). "Immortalized cells and one oncogene in malignant transformation: old insights on new explanation". BMC Cell Biology 12: 23. doi:10.1186/1471-2121-12-23. PMID 21605454. 
  2. ↑ 2.0 2.1 "Moral Guidance on Using COVID-19 Vaccines Developed with Human Fetal Cell Lines". Witherspoon Institute. May 25, 2020. https://www.thepublicdiscourse.com/2020/05/63752/. ""I received an e-mail a few months ago from Professor Frank Graham, who established this cell line. He tells me that to the best of his knowledge, the exact origin of the HEK293 fetal cells is unclear. They could have come from either a spontaneous miscarriage or an elective abortion."" 
  3. ↑ "USA FDA CTR For Biologics Evaluation and Research Vaccines and Related Biological Products Advisory Committee Meeting". Lines 14–22: USFDA. p. 81. https://www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_01.pdf. 
  4. ↑ "Characteristics of a human cell line transformed by DNA from human adenovirus type 5". The Journal of General Virology 36 (1): 59–74. July 1977. doi:10.1099/0022-1317-36-1-59. PMID 886304. 
  5. ↑ "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line". Virology 233 (2): 423–9. July 1997. doi:10.1006/viro.1997.8597. PMID 9217065. 
  6. ↑ 6.0 6.1 "Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells". FASEB Journal 16 (8): 869–71. June 2002. doi:10.1096/fj.01-0995fje. PMID 11967234. 
  7. ↑ 7.0 7.1 "Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations". Nature Communications 5 (8): 4767. September 2014. doi:10.1038/ncomms5767. PMID 25182477. Bibcode: 2014NatCo...5.4767L. 
  8. ↑ "ECACC Catalogue Entry for HEK 293". ECACC. http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85120602&collection=ecacc_gc. 
  9. ↑ "Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system". Mol. Cell. Biol. 7 (1): 379–387. Jan 1987. doi:10.1128/MCB.7.1.379. PMID 3031469. 
  10. ↑ "Production of high-titer helper-free retroviruses by transient transfection". Proc. Natl. Acad. Sci. USA 90 (18): 8392–8396. 15 Sep 1993. doi:10.1073/pnas.90.18.8392. PMID 7690960. Bibcode: 1993PNAS...90.8392P. 
  11. ↑ "A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction". Science 227 (4682): 23–28. 4 Jan 1985. doi:10.1126/science.2981116. PMID 2981116. Bibcode: 1985Sci...227...23R. 
  12. ↑ "Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations". Nat. Commun. 5: 4767. 3 Sep 2014. doi:10.1038/ncomms5767. PMID 25182477. Bibcode: 2014NatCo...5.4767L. 
  13. ↑ "Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action". British Journal of Pharmacology 148 (1): 16–24. May 2006. doi:10.1038/sj.bjp.0706709. PMID 16520744. 
  14. ↑ "Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter". Nucleic Acids Research 34 (5): e37. March 2006. doi:10.1093/nar/gkl034. PMID 16522642. 
  15. ↑ "The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site". Journal of Lipid Research 47 (6): 1146–56. June 2006. doi:10.1194/jlr.M500329-JLR200. PMID 16520488. 
  16. ↑ "Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism". Biochemistry 45 (10): 3125–33. March 2006. doi:10.1021/bi051634r. PMID 16519507. 
  17. ↑ "Identification of a functional nuclear export signal in the green fluorescent protein asFP499". Biochemical and Biophysical Research Communications 342 (4): 1178–82. April 2006. doi:10.1016/j.bbrc.2006.02.077. PMID 16516151. 
  18. ↑ "Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells". Molecular and Cellular Biology 5 (8): 2051–60. August 1985. doi:10.1128/mcb.5.8.2051. PMID 3018548. 
  19. ↑ "HEK293 cell line: a vehicle for the expression of recombinant proteins". Journal of Pharmacological and Toxicological Methods 51 (3): 187–200. 2005. doi:10.1016/j.vascn.2004.08.014. PMID 15862464. 
  20. ↑ "A simplified system for generating recombinant adenoviruses". Proc Natl Acad Sci U S A 95 (5): 2509–14. March 1998. doi:10.1073/pnas.95.5.2509. PMID 9482916. Bibcode: 1998PNAS...95.2509H. 
  21. ↑ Kovesdi, I; Hedley, SJ (August 2010). "Adenoviral producer cells.". Viruses 2 (8): 1681–703. doi:10.3390/v2081681. PMID 21994701. 
  22. ↑ "HEK293 Cell Line: human embryonic kidney cells". 2016. http://www.hek293.com/. 
  23. ↑ "Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells". European Journal of Pharmacology 390 (1–2): 51–9. February 2000. doi:10.1016/S0014-2999(99)00915-2. PMID 10708706. 
  24. ↑ "Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors". European Journal of Pharmacology 414 (2–3): 145–54. March 2001. doi:10.1016/S0014-2999(01)00789-0. PMID 11239914. 
  25. ↑ "M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms". Molecular Pharmacology 74 (2): 338–47. August 2008. doi:10.1124/mol.107.044750. PMID 18388243. 
  26. ↑ "Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells". The Journal of Biological Chemistry 280 (33): 29559–69. August 2005. doi:10.1074/jbc.M505842200. PMID 15972814. 
  27. ↑ "The ethics of HEK 293". The National Catholic Bioethics Quarterly 6 (3): 473–95. 2006. doi:10.5840/ncbq20066331. PMID 17091554. 
  28. ↑ 28.0 28.1 "The Facts about the COVID Vaccines and Fetal Cell Lines". National Review. 20 December 2020. https://www.nationalreview.com/2020/12/the-facts-about-the-pfizer-and-moderna-vaccines-and-fetal-cell-lines/. 
  29. ↑ "Balkan Insight: Tulpina religioasă a anti-vaccinismului se răspândește în Europa Centrală și de Est - International - HotNews.ro". 20 January 2021. https://www.hotnews.ro/stiri-international-24550811-balkan-insight-tulpina-religioasa-anti-vaccinismului-raspandeste-europa-centrala-est.htm. 
  30. ↑ RELIGIOUS STRAIN OF ANTI-VAX GROWS IN CEE, balkaninsight.com
  31. ↑ "Note on the morality of using some anti-Covid-19 vaccines.". Press Office of the Holy See. 21 December 2020. https://press.vatican.va/content/salastampa/it/bollettino/pubblico/2020/12/21/0681/01591.html#ing. 
  32. ↑ "There are no foetal cells in the AstraZeneca Covid-19 vaccine". Full Fact. 26 November 2020. https://fullfact.org/online/foetal-cells-covid-vaccine/. 
  33. ↑ Regalado, Antonio (7 October 2020). "Trump's antibody treatment was tested using cells originally derived from an abortion" (in en). https://www.technologyreview.com/2020/10/07/1009664/trumps-antibody-treatment-was-tested-using-cells-from-an-abortion/. ""It's how you want to parse it," says Alexandra Bowie, a Regeneron spokesperson. "But the 293T cell lines available today are not considered fetal tissue"" 

External links



Retrieved from "https://handwiki.org/wiki/index.php?title=Biology:HEK_293_cells&oldid=3624151"