A HEK293 Cell Database- put together by Gerry Shaw

Introduction

    HEK 293 cells were generated by transformation of human embryonic kidney cell cultures (hence HEK) with sheared adenovirus 5 DNA, and were first described in 1977 (Graham et al., J. Gen Virol 1977 Jul;36(1):59-74). The story of this cell line is quite an interesting one, getting to the very roots of modern molecular biology. Frank Graham was working with Alex Van der Eb in the University of Leiden in Holland, and developed the calcium phosphate method of transfecting eukaryotic cells, an astonishing technique described in a classic paper (Graham and Van der Eb. Virology 1973 Apr;52(2):456-67); who would have thought that you would only have to mix DNA with a bit of calcium phosphate to get the DNA incorporated and even expressed in mammalian cells? The story of this discovery was described by Graham himself for Current Contents in 1988 (link to *.pdf here). By 1977 Graham had moved to Canada, and from there produced the other classical paper describing the derivation of the HEK 293 cells. There is also an interesting Current Contents article by Graham describing this work (link to *.pdf here). Also, check out the transcript of an FDA meeting entitled "Vaccines and related biological products advisory committee" which took place in May 2001. At this meeting Alex Van der Eb describes the work that led to the HEK 293 derivation. I was particularly fascinated that he actually speculated that these cells may have originated from a rare neuronal cell in the kidney cell cultures, since at about the same time but completely independently we had come to a similar conclusion (link to *.pdf here - Dr. Van der Eb's part starts on page 77, and the specific comment about the potential neuronal origin of 293 cells is on page 85).

    Presented here are a summary of the initial results obtained by DNA microarray analysis of 293 cells. This analysis was stimulated by the observation that 293 cells stain strongly and specifically with antibodies to several neurofilament proteins, which are generally thought of as excellent markers for neuronal lineage cells. As with quite a lot of interesting stories in Science this was as a result of a completely unexpected finding; I was making monoclonal antibodies to the beta-adrenergic receptor kinase 1 (beta-ARK), which is supposed to be endogenously heavily expressed in 293 cells. I was at the point of screening by immunocytochemistry on fixed 293 cells, which I had bumed from another lab. The staining pattern was likely to be weak and diffuse at this stage so I needed a good control. So I reached into the fridge for a monoclonal antibody which would have no business staining a kidney cell, and since one of the things I work on is neurofilaments, I got out one of our monoclonals to NF-M, one of the major neurofilament subunits. I was surprised to find that the 293 cells all stained strongly and in a clearly filamentous pattern with this antibody. Since I had made and characterized the particular antibody myself I knew immediately that this was no artifact, these cells really must contain NF-M. Further biochemical and immunological experiments verified the presence of large amounts of the major neurofilament subunits NF-M and NF-L, and smaller amounts of alpha-internexin. Small amounts of NF-H were detected in a few cells, so that the four major neurofilament subunits were detected in 293 cell cultures. We also detected vimentin and the two primitive keratins K8 and K18, both immunocytochemically and by immunoblotting. The keratins are primitive in that they are the first expressed in development, so are characteristic of the earliest differentiating tissues of the body. Early differentiating neurons typically express NF-L, NF-M and alpha-internexin, but only express NF-H in large amounts later, as seen in 293. We did not detect desmin, GFAP, peripherin or any the high molecular weight keratin subunits. The pattern of expression is quite similar to that seen in PC12 and Ntera-2 cells, which also express a mix of neurofilament subunits and the two primitive keratins. So the 293 cells were similar in several respects to early differentiating neurons and to two well defined neuronal lineage cell lines.

    Further studies revealed that several other cell lines derived from human embryonic kidney cultures by adenovirus transformation also express neurofilaments, as did adenovirus derived cell lines from human embryonic retina, and the basic pattern of neurofilament expression was similar to that of 293 cells. While adenovirus efficiently generate cell lines from human embryonic retinal cultures, it has always been very difficult to generate lines from human embryonic kidney cultures. We hypothesize that a rare neuronal lineage cell type is the target for adenovirus transformation in these kidney cultures, which would obviously explain why it is difficult to obtain these cells from embryonic kidney cultures but much easier from embryonic retinal cultures. Literature searches reveal that there are in fact a few neuronal lineage cells in developing mammalian kidney, which we propose are the preferred targets for adenovirus. Further literature searches suggest that adenoviruses tend to specifically transform neuronal lineage cells, in line with the hypothesis presented here. We conclude that 293 cells have a phenotype surprisingly similar to established neuronal lineage cell lines such as PC12 and Ntera-2 cells, and that adenoviruses target specifically neuronal lineage cells.

    Microarray analysis of 293 cells using Affymetrix and Clontech arrays revealed mRNA encoding many other proteins normally expressed in neuronal lineage cells, and the data is summarized here in Table 1. For access to one of the full data sets press here. Apologies to those of you who tried to access this in the last few months, there have been various problems associated with security measures on our local server- this link should work now, but you will have to select the "293.xls" file manually). The Affymetrix chips score in terms of "Average Difference", which is essentially the amount of hybridization detected on 16 different 25 mer specific probes specific for different regions of the mRNA to be tested. The hybridization signals obtained are compared to the signals from 16 different 25 mer control probes which are identical to the experimental probes except that they include a single deliberate base mismatch. The data is an Excel spreadsheet ordered by the average difference column, so that those mRNA producing the strongest signals are at the top.The highest average difference values were 100,000 and above, and correspond to ribosomal RNA, b-actin or other well known abundant RNA. The threshold of reliable detection is probably average difference values in the range of 500-1,000. In a few cases the Affymetrix chip has multiple probes for a particular mRNA. Where multiple probes are used the table reveals this by providing multiple average difference values, and also the "absolute call" value, which is "P" for present, "M" for maybe or "A" for absent. In some cases these results are in conflict and in these cases the data should be interpreted with caution. For specific questions email me at shaw@mbi.ufl.edu. Tables 2 - 7 proved a summary of data on mRNA encoding G protein coupled receptors, receptor tyrosine kinases and other important families of molecules. The mRNA indicated with a * in Table 1 were derived from Clontech cDNA arrays which do not use the average difference measurement. You can download a *.pdf of these findings as published in Faseb Journal Express here.

Table 1: mRNA detected in 293 cells normally expressed specifically in neuronal cells

Accession

mRNA

Average Difference

X56009

GSA, a subunit of trimeric G protein heavily expressed in brain

58,306.5

AF039656

Neuronal tissue-enriched acidic protein (NAP-22)

39,329.5

D78577

Brain specific 14-3-3 protein eta chain

32,977.8

Y07969

APRIL, a protein rich in leucine residues heavily expressed in brain

32,381

AJ002308

Homo sapiens mRNA for synaptogyrin 2

22,737.3

M20471

Human brain-type clathrin light-chain a transcript

21,077.8

S65738

Human ADF (actin depolymerizing factor) particularly abundant in brain

17,101.8

AF053641

CSE1, brain specific isotype of cellular apoptosis susceptibility protein 1

12,396.7

X55110

MK a brain developmentally regulated neurite outgrowth-promoting protein

11,394.1

X05608

NF-L, neurofilament light chain, neuron specific class IV intermediate filament protein

9,185.4

S80071

Human brain-specific L-proline transporter

8,389.6

U64520

Human synaptobrevin-3 mRNA

7,767.4

X51956

Neuron specific enolase

6,135.3

S78296

a-internexin/neurofilament-66, neuron specific class IV intermediate filament protein

5,635.5

X99076

NRGN, human neurogranin gene, expressed in brain associated with synaptic vesicles

4,594.3

Y00067

NF-M, neurofilament medium chain, neuron specific class IV intermediate filament protein

4,317

AF042792

Homo sapiens alpha 2 delta calcium channel subunit isoform I

3916.8

U47025

Human fetal brain specific glycogen phosphorylase

3,853.3

AJ004832

Neuropathy target esterase, homologous to Drosophila neurodegeneration-associated protein

3,814.6

U57627

OCRL1, gene defective in Lowe oculocerebrorenal syndrome, expressed in brain and kidney.

3,676.1

AF059274

Neuroglycan C, a neural tissue-specific transmembrane chondroitin sulfate proteoglycan

3,248

U40998

HRG4, human retinal gene 4, expressed in retinal photoreceptors and not it kidney.

3,220.3

U07364

Human inwardly rectifying potassium channel mRNA,

3148.6

M67468

FMR-1, fragile X mental retardation protein expressed in brain

2,902.5

U66406

EPH-related PTK receptor ligand LERK-8 (Eplg8), restricted to brain in adult

2,756.4

S73591

VDUP1 (vitamin D upregulated protein) a brain-expressed homologue of HHCPA78

2,602.7

M20469

Human brain-type clathrin light-chain b transcript

2,343.7

AF015767

BRE, brain and reproductive organ-expressed protein

2,220.5

U61849

Human neuronal pentraxin 1 (NPTX1)

2,100.4

U49089

NE-dlg human neuronal and endocrine homologue of Drosophila Discs Large (dlg).

2,059.9

U87947

HNMP-1, hematopoietic and neural membrane protein differentially regulated in neural development and injury.

2,039.9

X02404

Calcitonin gene related polypeptide, second gene product

1,985.7

AF025304

Protein-tyrosine kinase EPHB2v (EPHB2), heavily expressed in brain

1,985.1

M55513

Human potassium channel (HPCN1) mRNA

1,712.5

D89016

Member of the Rho/Rac GEF Family associated with human neuroblastoma:

1,694.9

X70297

Human alpha 7 acetylcholine receptor:

1,663

U78181

Human sodium channel 2 (hBNaC2) mRNA

1,623

U07139

Human voltage-gated calcium channel beta subunit mRNA

1,577.8

AJ011733

Homo sapiens mRNA for synaptogyrin 4 protein

1,060.4

AF078544

BMCP (Brain mitochondrial carrier protein) 1, a mitochondrial carrier with high expression in the central nervous

994.2

D83699

DP5, neuron specific gene capable of inducing neuronal cell death

841

M98528

21kDa Golgi associated human neuron specific protein, homologous to mouse p1A75

785.7

U69883

Human calcium-activated potassium channel hSK1 (SK) mRNA

763

AJ002309

Homo sapiens mRNA for synaptogyrin 3

715.3

U57911

239FB mRNA, expressed in fetal human brain but not detectable in other fetal tissues

655.5

AF039945

Homo sapiens synaptojanin 2B mRNA

630.1

M55047

Human synaptotagmin mRNA

623.7

L10333

NSP, Neuroendocrine specific protein A, of molecular weight 135kDa

607.9

S82769

GABAA receptor gamma 3 subunit, fetal brain

576.5

AB003592

NB-3, a neuronal specific member of the contactin/F3 subgroup of the IgG family of adhesion molecule

499.7

M55514

Human potassium channel (HPCN2) mRNA

478.1

U58522

Human huntingtin interacting protein (HIP2), a ubiquitin conjugating enzyme heavily expressed in brain

446.1

AF058718

Putative 13 S Golgi transport complex 90kD subunit brain-specific isoform

434.1

U77970

NPAS2, a basic helix-loop-helix transcription factor expressed only in the nervous system

369.5

M76559

Human neuronal DHP-sensitive, voltage-dependent, calcium channel alpha-2b

352.3

U29195

Human neuronal pentraxin II (NPTX2)

230.1

H40164*

Human homologue of brain specific polypeptide pep-19

ND

R35139*

Human homologue of brain specific transcription factor BRN-5

ND

H41862*

Human homologue of hippocalcin, a brain specific recoverin family calcium binding protein

ND

H13022*

Human:neuronal acetylcholine receptor protein, alpha-5

ND

BC008730*

Brain specific hexokinase

ND

Table 2: G Protein coupled receptors

Accession

Name

Average Difference

X80818:

Metabotropic glutamate receptor type 4

10,779.1

AF027957:

Orphan receptor GPR35

5,831

X65633:

Adrenocorticotropic hormone (ACTH) receptor

4,071

AF062006:

Orphan receptor HG38

  3,780

AF011466:

Lysophosphatidic acid receptor Edg-4

  3,520.3

AJ225028:

GABA-B R1a receptor

  3,093.3

AF091890:

G-protein coupled receptor RE2

8,006.4, 2,805.8 (A, P)

U68487:

5-hydroxytryptamine 7 receptor isoform b

  2,629

L06797:

Clone L5 orphan receptor

  2,185.4

D16827:

Fifth somatostatin receptor subtype

  2,177.3

U56421:

Olfactory receptor (OLF3) gene,

  2,097.2

J03853:

Alpha-2-adrenergic receptor

  2,096.4

M81590:

Serotonin 1D receptor (5-HT1D~)

  1,930.7

U31216:

Metabotropic glutamate receptor 1 beta (mGluR1beta)

  1,925.6

Y10148:

Neurotensin NTR2 receptor

  1,868

U34038:

Proteinase-activated receptor-2

1,590.5, 1,333.6 (P, P)

M84562:

Formyl peptide receptor-like receptor (FPRL1)

  1,316.9

M62424:

Thrombin receptor

  1,226.7

AF095448:

G protein-coupled receptor (RAIG1)

  1,166.6

X68487:

A2b adenosine receptor

  1,058.8

X95876:

Orphan G-protein coupled receptor

  1,054.5

X51362:

Dopamine D2

  973.1

D17516:

Pituitary adenylate cyclase activating polypeptide receptor

   648.8

U64871:

Putative G protein-coupled receptor (GPR19)

   634.9

M15169:

Human beta-2-adrenergic receptor

    579.3

U29589:

M3 muscarinic acetylcholine receptor (CHRM3) gene

    517.6

U13666:

Orphan G protein-coupled receptor (GPR1) gene

    444.2

X86163:

B2-bradykinin receptor

422.8

Table 3: TGFb receptor family

Accession

Name

Average Difference

X77533:

Activin type II receptor

3,847.5

D31770:

Activin type II A receptor

723.5

L07594

TGF-beta type III receptor

735.2

D50683:

TGF-betaIIR alpha

459

AF054598:

TGF-beta type I receptor (TGFBR1) gene

455

Table 4: Cytokine Receptors

Accession

Name

Average Difference

AF063605

leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

4,446.6

U19247:

Interferon-gamma receptor alpha chain

2,110.1

X52425:

Interleukin 4 receptor

1,122.7

U32324:

Interleukin-11 receptor alpha chain

1,102.4

M29696:

Interleukin-7 receptor (IL-7)

 851

L42243:

Alternatively spliced interferon receptor (IFNAR2)

1,565.2, 793.6, 537.1, 267.4 (P, P, A, P)

M60459 :

Erythropoietin receptor mRNA, complete cds

1,396.5, 734.5 (P, P)

U60805:

Human oncostatin-M specific receptor beta subunit

 595.9, 221.3 (P, M)

X61615

Leukemia inhibitory factor (LIF) receptor.

 388.2

Table 5: Apoptosis related

Accession

Name

Average Difference

AF016266:

TRAIL receptor 2 mRNA

17,035.7

M58286:

Tumor necrosis factor receptor

  4,846.7

U83598:

Death domain receptor 3 soluble form (DDR3)

  2,503.1, 458.9 (P, P)

M14764:

Low affinity Nerve growth factor receptor

  916

Z75190:

Apolipoprotein E receptor 2

  721.2

Table 6: Ligand gated ion channel molecules

Accession

Name

Average Difference

X55019:

Acetylcholine receptor delta subunit

1,745.1

X70297:

Neuronal nicotinic acetylcholine receptor alpha-7

1,663

U33267:

Glycine receptor beta subunit (GLRB)

1,409.7

Y09765:

Putative GABA receptor epsilon subunit

1,298.6

U62434:

Acetylcholine receptor alpha5 subunit

1,010.8

X82068:

Glutamate receptor subunit GluRC

860.4

S82769:

GABAA receptor gamma 3 subunit

576.5

Table 7: Insulin related molecules

Accession

Name

Average Difference

U96876

Homo sapiens insulin induced protein 1 (INSIG1)

5,786.2

HSU08316

Human insulin-stimulated protein kinase 1 (ISPK-1)

2,815

Y00285:

Insulin-like growth factor II receptor

2,476.5

X81832:

Glucose-dependant insulinotropic polypeptide receptor

2,069.1

X04434:

Insulin-like growth factor I receptor

2,140.8, 237.9 (P, A)

S37730

Insulin-like growth factor binding protein 2

2,015.3

AF007567

Homo sapiens insulin receptor substrate 4

1,480

U20982

Insulin-like growth factor binding protein 4

1,029.8

X02160

Human mRNA for insulin receptor precursor

  810.2