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.
Similar content being viewed by others
References
Baxter RC (1994) Insulin-like growth factor binding proteins in the human circulation. Horm. Res. 42(4–5): 140–144
Bellingham DL, Sar M and Cidlowski JA (1992) Methotrexate-induced overexpression of functional glucocorticoid receptors in chinese hamster ovary cells. Mol. Cell. Endocrinol. 83(2–3): 153–171.
Baxter RC (1994) Insulin-like growth factor binding proteins in the human circulation. Horm. Res. 42(4–5): 140–144
Clark DG, Tomas FM, Withers RT, Brinkman M, Berry MN, Oliver JR, Owens PC, Butler RN, Ballard FJ and Nestel PJ (1995) Differences in substrate metabolism between sel-perceived ‘large-eating’ and "small-eating’ women. Int. J. Obes. Relat. Metab. Disord. 19(4): 245–252
Clemmons DR (1992) IGF binding proteins: regulation of cellular actions. Growth Regulation 2: 80–87.
Fisher WE, Boros LG, O'Dorisio MS and Schirmer WJ (1995) GI hormonal changes in diabetes influence pancreatic cancer growth. J. Surg. Res. 58(6): 754–758
Gu MB, Kern JA, Todd P and Kompala DS (1992) Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells. Cytotechnology 9: 237–245.
Kane SE, Reinhard DH, Fordis CM, Pastan I and Gottesman MM (1989) A new vector using humanmultidrug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells. Gene 84: 439–446.
Kaufman RJ, Murtha P, Ingolia DE, Yeung CY and Kellems RE (1986) Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells. Proc. Natl. Acad. Sci. USA 83(10): 3136–40.
Kaufman RJ, Wasley LC, Spiliotes AJ, Gossels SD, Latt SA, Larsen GR and Kay RM (1985) Coamplification and coexpression of human tissue-type Plasminogen Activator and murine Dihydro-folate Reductase Sequences in Chinese Hamster Ovary Cells. Mol. Cell. Biol. 5(7): 1750–59.
Kemball-Cook G, Garner I, Imanaka Y, Nishimura T, O'Brien DP, Tuddenham EG and McVey JH (1994) High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector. Gene 139(2): 275–279.
Kolaczynski JW and Caro JF (1994) Insulin-like growth factor-1 therapy in diabetes: physiologic basis, clinical benefits and risks. Ann. Intern. Med. 120(1): 47–55.
Lee PD, Conover CA and Powell DR (1993) Regulation and function of insulin-like growth factor binding protein-1. Proc. Soc. Exp. Biol. Med. 204(1): 4–29.
Ljung C, Bastrup B, Hansson L, Schenström K, Hartmanis M, Mellström K, Hall K, Wiwall I-L and Lake M (1990) Cloning, expression and purification of IGF-Binding Protein, BP-1. 5th European Congress on Biotechnology in Copenhagen. Proceedings 2: 728–730.
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 and Jörnvall H (1989) Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning. Eur. J. Biochem. 180: 259–265.
McCormick F, Trahey M, Innis M, Dieckmann B and Ringold G (1984) Inducible expression of amplified human beta interferon genes in CHO cells. Mol. Cell. Biol. 4: 166–172.
Page MJ (1985) Expression of amplified human beta interferon genes using heavy metal induction in Chinese Hamster Ovary cells. Gene 37: 139–144.
Rasmussen MH, Frystyk J, Andersen T, Breum L, Christiansen JS and Hilsted J (1994) 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 43(3): 315–319.
Sanders PG, Hussein A, Coggins L and Wilson R (1987) Gene amplification: the Chinese hamster glutamine synthetase gene. Dev. Biol. Stand. 66: 55–63.
Schacht E (1994) Differential therapy of osteoporosis — An overview based on recent findings regarding the pathogenesis. Z. Rheumatol. 53(5): 274–298.
Sirotnak FM, Moccio DM, Kelleher LE and Goutas LJ (1981) Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1200 clonal cell lines derived in vivo. Cancer res. 41: 4447–4452.
Urlaub G and Chasin LA (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77: 4216–4220.
Varshavsky A (1981) Phorbol ester dramatically increases incidence of methotrexate-resistant colony-forming mouse cells: possible mechanisms and relevance to tumor promotion. Cell 25: 561–572.
Rights and permissions
About this article
Cite this article
Dyring, C., Mellström, K. Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells. Cytotechnology 24, 193–200 (1997). https://doi.org/10.1023/A:1007999512662
Issue Date:
DOI: https://doi.org/10.1023/A:1007999512662