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. 1997 Sep;24(3):193–200. doi: 10.1023/A:1007999512662

Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells

Charlotte Dyring, Karin Mellström
PMCID: PMC3449622  PMID: 22358762

Abstract

Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production.

Keywords: CHO cells, DHFR, IGFBP-1, stability

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References

  1. Baxter RC. Insulin-like growth factor binding proteins in the human circulation. Horm. Res. 1994;42(4-5):140–144. doi: 10.1159/000184186. [DOI] [PubMed] [Google Scholar]
  2. Bellingham DL, Sar M, Cidlowski JA. Methotrexate-induced overexpression of functional glucocorticoid receptors in chinese hamster ovary cells. Mol. Cell. Endocrinol. 1992;83(2-3):153–171. doi: 10.1016/0303-7207(92)90156-Z. [DOI] [PubMed] [Google Scholar]
  3. Baxter RC. Insulin-like growth factor binding proteins in the human circulation. Horm. Res. 1994;42(4-5):140–144. doi: 10.1159/000184186. [DOI] [PubMed] [Google Scholar]
  4. Clark DG, Tomas FM, Withers RT, Brinkman M, Berry MN, Oliver JR, Owens PC, Butler RN, Ballard FJ, Nestel PJ. Differences in substrate metabolism between sel-perceived ‘large-eating’ and ‘small-eating’ women. Int. J. Obes. Relat. Metab. Disord. 1995;19(4):245–252. [PubMed] [Google Scholar]
  5. Clemmons DR. IGF binding proteins: regulation of cellular actions. Growth Regulation. 1992;2:80–87. [PubMed] [Google Scholar]
  6. Fisher WE, Boros LG, O'Dorisio MS, Schirmer WJ. GI hormonal changes in diabetes influence pancreatic cancer growth. J. Surg. Res. 1995;58(6):754–758. doi: 10.1006/jsre.1995.1119. [DOI] [PubMed] [Google Scholar]
  7. Gu MB, Kern JA, Todd P, Kompala DS. Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells. Cytotechnology. 1992;9:237–245. doi: 10.1007/BF02521751. [DOI] [PubMed] [Google Scholar]
  8. Kane SE, Reinhard DH, Fordis CM, Pastan I, Gottesman MM. A new vector using humanmultidrug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells. Gene. 1989;84:439–446. doi: 10.1016/0378-1119(89)90518-0. [DOI] [PubMed] [Google Scholar]
  9. Kaufman RJ, Murtha P, Ingolia DE, Yeung CY, Kellems RE. Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells. Proc. Natl. Acad. Sci. USA. 1986;83(10):3136–40. doi: 10.1073/pnas.83.10.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaufman RJ, Wasley LC, Spiliotes AJ, Gossels SD, Latt SA, Larsen GR, Kay RM. Coamplification and coexpression of human tissue-type Plasminogen Activator and murine Dihydro-folate Reductase Sequences in Chinese Hamster Ovary Cells. Mol. Cell. Biol. 1985;5(7):1750–59. doi: 10.1128/mcb.5.7.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kemball-Cook G, Garner I, Imanaka Y, Nishimura T, O'Brien DP, Tuddenham EG, McVey JH. High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector. Gene. 1994;139(2):275–279. doi: 10.1016/0378-1119(94)90769-2. [DOI] [PubMed] [Google Scholar]
  12. Kolaczynski JW, Caro JF. Insulin-like growth factor-1 therapy in diabetes: physiologic basis, clinical benefits and risks. Ann. Intern. Med. 1994;120(1):47–55. doi: 10.7326/0003-4819-120-1-199401010-00009. [DOI] [PubMed] [Google Scholar]
  13. Lee PD, Conover CA, Powell DR. Regulation and function of insulin-like growth factor binding protein-1. Proc. Soc. Exp. Biol. Med. 1993;204(1):4–29. doi: 10.3181/00379727-204-43630. [DOI] [PubMed] [Google Scholar]
  14. Ljung C, Bastrup B, Hansson L, Schenström K, Hartmanis M, Mellström K, Hall K, Wiwall I-L, Lake M. Cloning, expression and purification of IGF-Binding Protein, BP-1. 5th European Congress on Biotechnology in Copenhagen. Proceedings. 1990;2:728–730. [Google Scholar]
  15. Luthman H, Söderling-Barros J, Persson B, Engberg C, Stern I, Lake M, Franzén S, Israelsson M, Rådén B, Lindgren B, Hjelmqvist L, Enerbäck S, Carlsson P, Bjursell G, Póvoa G, Hall K, Jörnvall H. Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning. Eur. J. Biochem. 1989;180:259–265. doi: 10.1111/j.1432-1033.1989.tb14641.x. [DOI] [PubMed] [Google Scholar]
  16. McCormick F, Trahey M, Innis M, Dieckmann B, Ringold G. Inducible expression of amplified human beta interferon genes in CHO cells. Mol. Cell. Biol. 1984;4:166–172. doi: 10.1128/mcb.4.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Page MJ. Expression of amplified human beta interferon genes using heavy metal induction in Chinese Hamster Ovary cells. Gene. 1985;37:139–144. doi: 10.1016/0378-1119(85)90266-5. [DOI] [PubMed] [Google Scholar]
  18. Rasmussen MH, Frystyk J, Andersen T, Breum L, Christiansen JS, Hilsted J. The impact of obesity, fat distribution, and energy restriction on insulin-like growth factor-1 (IGF-1), IGF-binding protein-3, insulin and growth hormone. Metabolism. 1994;43(3):315–319. doi: 10.1016/0026-0495(94)90099-X. [DOI] [PubMed] [Google Scholar]
  19. Sanders PG, Hussein A, Coggins L, Wilson R. Gene amplification: the Chinese hamster glutamine synthetase gene. Dev. Biol. Stand. 1987;66:55–63. [PubMed] [Google Scholar]
  20. Schacht E. Differential therapy of osteoporosis — An overview based on recent findings regarding the pathogenesis. Z. Rheumatol. 1994;53(5):274–298. [PubMed] [Google Scholar]
  21. Sirotnak FM, Moccio DM, Kelleher LE, Goutas LJ. Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1200 clonal cell lines derived in vivo. Cancer res. 1981;41:4447–4452. [PubMed] [Google Scholar]
  22. Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA. 1980;77:4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Varshavsky A. Phorbol ester dramatically increases incidence of methotrexate-resistant colony-forming mouse cells: possible mechanisms and relevance to tumor promotion. Cell. 1981;25:561–572. doi: 10.1016/0092-8674(81)90074-X. [DOI] [PubMed] [Google Scholar]

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