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Background and explanation

Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations

Yao-Cheng Lin, Morgane Boone, Leander Meuris, Irma Lemmens, Nadine Van Roy, Arne Soete, Joke Reumers, Matthieu Moisse, Stéphane Plaisance, Radoje Drmanac, Jason Chen, Frank Speleman, Diether Lambrechts, Yves Van de Peer, Jan Tavernier & Nico Callewaert

The popular Human Embryonic Kidney (HEK) 293 cell line and its derivatives have been used for decades in both basic and applied research, for purposes ranging from signal transduction and protein interaction studies, over viral packaging, to rapid small-scale protein expression for structural biology and recombinant pharmaceutical production.

The original 293 cell line was derived in 1973, from the kidney of an aborted human embryo of unknown parenthood (Graham, 1992; Graham et al., 1977). Earlier experiments showed that rodent embryonic kidney cells could successfully be immortalized with sheared Adenovirus 5 (Ad5) DNA. On the contrary, human embryonic kidney cell transformation proved to be difficult, and it required multiple (293!) rounds of tedious experimentation before a single, albeit slow growing clone was obtained. This clone underwent a cultivation crisis that is reported to have lasted several months, after which the fast-growing derivative cells were finally established as HEK293 or 293 cells (Graham, 1992). The recalcitrance to transformation suggests that the cells that could be transformed by Ad5 DNA in human embryonic kidney must be very rare. This resistance to transformation with Ad5 DNA is also true for many other human tissues, except for the human retina, which was used to derive the Per.C6 cell line. Taken together with microarray data showing expression of a multitude of neuron-specific genes (Shaw et al., 2002), 293 cells are believed to have originated from an immature neuronal cell in the embryonic kidney. As for the Ad5 DNA, a 4 kbp adenoviral genome fragment including the E1A/E1B gene is known to have integrated in chromosome 19 (Louis et al., 1997). Cytogenetic analysis of the 293 line revealed multiple and complex chromosomal abnormalities (Bylund et al., 2004).

The most frequently used 293 derivative is the 293T line, which expresses a temperature sensitive allele of the SV40 T antigen (Rio et al., 1985; DuBridge et al., 1987). Vectors containing an SV40 ori can thus be amplified, which considerably increases the expression levels obtained with transient transfection.

In 1984, the original 293 line was adapted to suspension growth through serial passaging in Joklik’s modified minimal Eagle’s medium (Stillman and Gluzman, 1985). It took about 7 months of passaging to fully adapt the cells; the first passages were very difficult and the few cells that grew through are likely to have been almost clonal (Dr. Bruce Stillman, personal communication). The fully adapted cell line is known as 293S and has given rise to what we refer to as the 293SG line. These cells, selected for resistance against the Ricin toxin after ethylmethanesulfonate (EMS) mutagenesis, lack N-acetylglucosaminyltransferase I activity (encoded by the MGAT1 gene) and accordingly predominantly modify glycoproteins with the Man5GlcNAc2 N-glycan. They additionally express the tetR repressor, enabling tetracyclin-inducible protein expression (Reeves et al., 2002), and are therefore well-suited (and widely used) for the production of homogeneously N-glycosylated proteins.

The final two 293-derived lines included in this resource were developed for protein-protein interaction screening (293FTM) and glyco-engineering (293SGGD) purposes. Any details on their generation can be found in the Supplementary Information section of the genome paper and references therein.

The HEK293 Multigenome Variation Viewer was built for sequence- and copy number variation browsing of these 6 cell biology ‘workhorse’ cell lines. This should not only improve our understanding of basic 293 cell line biology, but also prove to be a great advantage for 293 cell engineering through sequence-specific genome editing enzymes (Doyon et al., 2011; Hockemeyer et al., 2011) and genome-wide siRNA (Root et al., 2006; Coussens et al., 2011). All sequenced clones are made available to the community as well. We refer to the original 293 genome paper for a more in-depth discussion of the data and some remarkable results.

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Coussens et al. (2011), J.Vis.Exp. 58, 3305
Doyon et al. (2011), Nat. Cell Biol. 13, 331-337
DuBridge et al. (1987), Mol. Cell Biol. 7, 379-387
Graham (1992),Current Contents/Life Sciences 8, 8
Graham et al. (1977), J. Gen.Virol. 36, 59-72
Hockemeyer et al. (2011), Nat. Biotechnol. 29, 731-734
Louis et al. (1997), Virology 233, 423-429
Reeves et al. (2002), Proc. Natl. Acad. Sci. USA 99, 13419-13424
Rio et al. (1985), Science 277, 23-28
Root et al. (2006), Nat. Methods 3, 715-719
Shaw et al. (2002), FASEB J 16, 869
Stillman and Gluzman (1985), Mol. Cell Biol. 5, 2051-2060