, Volume 33, Issue 1–3, pp 139–145 | Cite as

Transient transfection induces different intracellular calcium signaling in CHO K1 versus HEK 293 cells

  • A. K. Preuss
  • J. A. Connor
  • H. Vogel


For the controlled production of recombinant proteinsin mammalian cells by transient transfection, it maybe desirable not only to manipulate, but also todiagnose the expression success early. Here, weapplied laser scanning confocal microscopy to monitortransfection induced intracellular Ca2+responses. We compared Chinese hamster ovary (CHO K1)versus human embryo kidney (HEK) 293 cell lines, whichdiffer largely in their transfectability. An improvedcalcium phosphate transfection method was used for itssimplicity and its demonstrated upscale potential.Cytosolic Ca2+ signaling appeared to inverselyreflect the cellular transfection fate. Virtually allCHO cells exhibited asynchronous, cytosolicCa2+ oscillations, which peaked 4 h afteraddition of the transfecting solution. Yet, most ofthe HEK cells displayed a slow and continuousCa2+ increase over the time of transfection. CHOcells, when exposed to a transfection-enhancingglycerol shock, strongly downregulated their Ca2+response, including its oscillations. When treatedwith thapsigargin, a Ca2+ store depleting drug,the number of successfully transfected CHO cells was significantly reduced. Our result points tointracellular store release as a critical componentfor the transfection fate of CHO cells, and its early detection before product visualization.

calcium phosphate CHO-K1 cytosolic calcium signaling HEK 293 laser scanning confocal microscopy transient transfection 


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  1. Bading H, Hardingham GE, Johnson CM and Chawla S (1997) Gene regulation by nuclear and cytoplasmic calcium signals. Biochem Biophys Res Commun 236: 541–543.Google Scholar
  2. Begum N, Leitner W, Reusch JE, Sussman KE and Draznin B (1993) GLUT-4 phosphorylation and its intrinsic activity. Mechanism of Ca(2+)-induced inhibition of insulin-stimulated glucose transport. J Biol Chem 268: 3352–3356.Google Scholar
  3. Berridge MJ (1997) Elementary and global aspects of calcium signalling. J Exp Biol 200: 315–319.Google Scholar
  4. Coco S, Verderio C, De Camilli P and Matteoli M (1998) Calcium dependence of synaptic vesicle recycling before and after synaptogenesis. J Neurochem 71: 1987–1992.Google Scholar
  5. Dolmetsch RE, Xu K and Lewis RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392: 933–936.Google Scholar
  6. Greber UF and Gerace L (1995) Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus. J Cell Biol 128: 5–14.Google Scholar
  7. Greber UF, Suomalainen M, Stidwill RP, Boucke K, Ebersold MW and Helenius A (1997) The role of the nuclear pore complex in adenovirus DNA entry. EMBO J 16: 5998–6007.Google Scholar
  8. Jordan M, Schallhorn A and Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24: 596–601.Google Scholar
  9. Loyter A, Scangos GA and Ruddle FH (1982) Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci USA 79: 422–426.Google Scholar
  10. Orrantia E, Li ZG and Chang PL (1990) Energy dependence of DNA-mediated gene transfer and expression. Somat Cell Mol Genet 16: 305–310.Google Scholar
  11. Palfrey HC and Nairn AC (1995) Calcium-dependent regulation of protein synthesis. Adv Second Messenger Phosphoprotein Res 30: 191–223.Google Scholar
  12. Parekh AB and Penner R (1997) Store depletion and calcium influx. Physiol Rev 77: 901–930.Google Scholar
  13. Perez-Terzic C, Jaconi M and Clapham DE (1997) Nuclear calcium and the regulation of the nuclear pore complex. Bioessays 19: 787–792.Google Scholar
  14. Preuss AK, Pick HM, Wurm F and Vogel H (1999) Transient transfection induces early cytosolic calcium signaling in CHO-K1 cells. In: Ikura K, Nagao M, Masuda S and Sasaki R (eds) Animal Cell Technology: Challenges for the 21st Century (pp. 17–21). Kluwer Academic Publishers, Dordrecht.Google Scholar
  15. Rosales C and Brown EJ (1992) Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate. J Biol Chem 267: 5265–5271.Google Scholar
  16. Rosen LB, Ginty DD and Greenberg ME (1995) Calcium regulation of gene expression. Adv Second Messenger Phosphoprotein Res 30: 225–253.Google Scholar
  17. Simonsen CC and McGrogan M (1994) The molecular biology of production cell lines. Biologicals 22: 85–94.Google Scholar
  18. Sompayrac LM and Danna KJ (1981) Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci USA 78, 7575–7578.Google Scholar
  19. Staedel C, Remy JS, Hua Z, Broker TR, Chow LT and Behr JP (1994) High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine: DNA complexes. J Invest Dermatol 102: 768–772.Google Scholar
  20. Stehno-Bittel L, Perez-Terzic C and Clapham DE (1995) Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2+ store. Science 270: 1835–1838.Google Scholar
  21. Wang HX, Ouyang M, Zhang WM, Sheng JZ and Wong TM (1998) Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte. Clin Exp Pharmacol Physiol 25: 257–265.Google Scholar
  22. Wei H and Perry DC (1996) Dantrolene is cytoprotective in two models of neuronal cell death. Neurochem 67: 2390–2398.Google Scholar
  23. Won JG and Orth DN (1995) Role of inositol trisphosphate-sensitive calcium stores in the regulation of adrenocorticotropin secretion by perifused rat anterior pituitary cells. Endocrinology 136: 5399–5408.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • A. K. Preuss
    • 1
  • J. A. Connor
    • 2
  • H. Vogel
    • 3
  1. 1.Laboratory of Physical Chemistry of Polymers and Membranes, Chemistry DepartmentSwiss Federal Institute of TechnologyLausanneSwitzerland
  2. 2.The Lovelace InstitutesAlbuquerqueU.S.A.
  3. 3.Laboratory of Physical Chemistry of Polymers and Membranes, Chemistry DepartmentSwiss Federal Institute of TechnologyLausanneSwitzerland

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