, Volume 24, Issue 1, pp 55–64 | Cite as

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

  • S. M. N. Hunt
  • S. C. O. Pak
  • M. W. Bridges
  • P. P. Gray
  • M. J. Sleigh


Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

CHO IGF-I serum-free autocrine growth cell culture 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ballard FJ, Francis GL, Ross M, Bagley CJ, May B and Wallace JC (1987) Natural and synthetic forms of insulin-like growth factor-1 (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding. Biochem. Biophys. Res. Commun. 149: 398–404.PubMedCrossRefGoogle Scholar
  2. Ballotti R, Nielsen FC, Pringle N, Kowalski A, Richardson WD, Van Obberghen E and Gammeltoft S (1987) Insulin-like growth factor I in cultured rat astrocytes: expression of the gene, and receptor tyrosine kinase. Embo. J. 6: 3633–3639.PubMedGoogle Scholar
  3. Barnes D and Sato G (1980) Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102: 255–270.PubMedCrossRefGoogle Scholar
  4. Baxter RC (1986) The somatomedins: insulin-like growth factors. Adv. Clin. Chem. 25: 49–115.PubMedGoogle Scholar
  5. Bayne ML, Applebaum J, Chicchi GG, Hayes NS, Green BG and Cascieri MA (1988) Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast. Gene 66: 235–244.PubMedCrossRefGoogle Scholar
  6. Bayne ML, Cascieri MA, kelder B, Applebaum J, Chicchi GG, Shapiro JA, Pasleau F and Kopchick JJ (1987) Expression of a synthetic gene encoding human insulin-like growth factor 1 in cultured mouse fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 84: 2638–2642.PubMedCrossRefGoogle Scholar
  7. Bendig MM (1988) The production of foreign proteins in mammalian cells. Genet. Eng. 7: 91–127.PubMedGoogle Scholar
  8. Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB and Bierich JR (1989) Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 125: 766–772.PubMedGoogle Scholar
  9. Bovenberg WA, Dauwerse JG, Pospiech HM, Van Buul Offers SC, Van den Brande JI and Sussenbach JS (1990) Expression of recombinant human insulin-like growth factor I in mammalian cells. Mol. Cell. Endocrinol. 74: 45–59.PubMedCrossRefGoogle Scholar
  10. Cascieri MA, Hayes NS, Kelder B, Kopchick JJ, Chicchi GG, Slater EE and Bayne ML (1988) Inability of a mouse cell line transformed to produce biologically active recombinant human insulin-like growth factor I (IGF-I) to respond to exogenously added IGF-I. Endocrinology 122: 1314–1320.PubMedGoogle Scholar
  11. Chen C and Okayama H (1988) Calcium phosphate-mediated gene transfer: A highly effective system for stably transforming cells with plasmid DNA. Bio/Techniques 6: 632–638.Google Scholar
  12. Clemmons DR (1984) Multiple hormones stimulate the production of somatomedin by cultured human fibroblasts. J. Clin. Endocrinol. Metab. 58: 850–856.PubMedCrossRefGoogle Scholar
  13. Clemmons DR and Shaw DS (1983) Variables controlling somatomedin production by cultured human fibroblasts. J. Cell. Physiol. 115: 137–142.PubMedCrossRefGoogle Scholar
  14. Clemmons DR, Underwood LE and Van Wyk JJ (1981) Hormonal control of immunoreactive somatomedin production by cultured human fibroblasts. J. Clin. Invest. 67: 10–19.PubMedCrossRefGoogle Scholar
  15. Dai Z, Stiles AD, Moats SB, Van Wyk JJ and D'Ercole AJ (1992a) Interaction of secreted insulin-like growth factor-I (IGF-I) with cell surface receptors is the dominant mechanism of IGF-I's autocrine actions. J. Biol. Chem. 267: 19565–19571.PubMedGoogle Scholar
  16. Dai Z, Takahashi SI, Van Wyk JJ and D'Ercole AJ (1992b) Creation of an autocrine model of insulin-like growth factor-I action in transfected FRTL-5 cells. Endocrinology 130: 3175–3183.PubMedCrossRefGoogle Scholar
  17. Daughaday WH and Rotwein P (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Rev. 10: 68–91.PubMedGoogle Scholar
  18. Friedman JS, Cofer CL, Anderson CL, Kushner JA, Gray PP, Chapman GE, Stuart MC, Lazarus L, Shine J and Kushner PJ (1989) High expression in mammalian cells without amplification. Bio/Technology 7: 359–362.CrossRefGoogle Scholar
  19. Friedman KD, Rosen NL, Newman PJ and Montgomery RR (1988) Enzymatic amplification of specific cDNA inserts from lambda gt11 libraries. Nucleic Acids Res. 16: 8718.PubMedGoogle Scholar
  20. Froesch ER, Schmid C, Schwander J and Zapf J (1985) Actions of insulin-like growth factors. Ann. Rev. Physiol. 443–467.Google Scholar
  21. Gough NM (1988) Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal. Biochem. 173: 93–95.PubMedCrossRefGoogle Scholar
  22. Jansen M, van Schaik FMA, Ricker AT, Bullock B, Woods DE, Gabbay KH, Nussbaum AL, Sussenbach JS and Van den Brande JL (1983) Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature 306: 609–611.PubMedCrossRefGoogle Scholar
  23. Kelly JL, Sanchez A, Brown GS, Chesterman CN and Sleigh MJ (1993) Accumulation of PDGF B and cell-binding forms of PDGF A in the extracellular matrix. J. Cell. Biol. 121: 1153–1163.PubMedCrossRefGoogle Scholar
  24. Kozak M (1986) Point Mutations Define a Sequence Flanking the AUG initiator Codon that Modulates Translation by Eukaryotic Ribosomes. Cell 44: 283–292.PubMedCrossRefGoogle Scholar
  25. Laemmli UK (1970) Cleavage of structural protein during assembly of the head bacteriophage T4. Nature 227: 680–685.PubMedCrossRefGoogle Scholar
  26. Laub O and Rutler WJ (1983) Expression of the human insulin gene and cDNA in a heterologous mammalian system. J. Biol. Chem. 258: 6043–6050.PubMedGoogle Scholar
  27. Maniatis T, Fritsch EF and Sambrook J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York.Google Scholar
  28. McKinnon P, Ross M, Wells JR, Ballard FJ and Francis GL (1991) Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1–3) IGF-I in Chinese hamster ovary cells. J. Mol. Endocrinol. 6: 231–239.PubMedCrossRefGoogle Scholar
  29. Moore HP, Walker MD, Lee F and Kelly RB (1983) Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell 35: 531–538.PubMedCrossRefGoogle Scholar
  30. Rechler MM and Nissley SP (1985) The nature and regulation of the receptors for insulin-like growth factors. Annu. Rev. Physiol. 47: 425–442.PubMedCrossRefGoogle Scholar
  31. Ross S and Englesberg E (1993) The competence progression model in CHO-K1 cells: the relationship between protein kinase C and immediate early gene expression in the insulin mitogenic signal. Biochim. Biophys. Acta 1177: 307–317.PubMedCrossRefGoogle Scholar
  32. Rotwein P, Pollock KM, Didier DK and Krivi GG (1986) Organization and sequence of the human insulin-like growth factor I gene. Alternative RNA processing produces two insulin-like growth factor I precursor peptides. J. Biol. Chem. 261: 4828–4832.PubMedGoogle Scholar
  33. Vollenweider F, Irminger JC, Gross DJ, Villa KL and Halban PA (1992) Processing of proinsulin by transfected hepatoma (FAO) cells. J. Biol. Chem. 267: 14629–14636.PubMedGoogle Scholar
  34. Vollenweider F, Irminger JC and Halban PA (1993) Substrate specificity of proinsulin conversion in the constitutive pathway of transfected FAO (hepatoma) cells. Diabetologia 36: 1322–1325.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • S. M. N. Hunt
    • 1
  • S. C. O. Pak
    • 1
  • M. W. Bridges
    • 1
  • P. P. Gray
    • 1
  • M. J. Sleigh
    • 2
  1. 1.Department of BiotechnologyUniversity of New South WalesKensingtonAustralia
  2. 2.Division of Biomolecular EngineeringCSIRONorth RydeAustralia

Personalised recommendations