Advertisement

Cytotechnology

, Volume 33, Issue 1–3, pp 37–46 | Cite as

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

  • Tomohiro Yoshikawa
  • Fumi Nakanishi
  • Seima Itami
  • Daisuke Kameoka
  • Takeshi OmasaEmail author
  • Yoshio Katakura
  • Michimasa Kishimoto
  • Ken-ichi Suga
Article

Abstract

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.

Chinese hamster ovary (CHO) dihydrofolate reductase (dhfr fluorescence in situ hybridization (FISH) gene amplification 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alt FW, Kellems RE, Bertino JR and Schimke RT (1978) Selective multiplication of dihydrofolate reductase genes in methotrexateresistant variants of cultured murine cells. J Biol Chem 253: 1357–1370.Google Scholar
  2. Assaraf YG and Schimke RT (1987) Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Natl Acad Sci USA 84: 7154–7158.Google Scholar
  3. Barclay BJ, Kunz BA, Little JG and Haynes RH (1982) Genetic and biochemical consequences of thymidylate stress. Can J Biochem 60: 172–184.Google Scholar
  4. Carroll SM, DeRose ML, Gaudray P, Moore CM, Needham-Vandevanter DR, von Hoff DD and Wahl GM (1988) Double minute chromosomes can be produced from precursors derived from a chromosomal deletion. Mol Cell Biol 8: 1525–1533.Google Scholar
  5. Chatterjee S and Price B (1991) Regression analysis by example. A Wiley-Interscience publication, pp. 1–21, New York, USA.Google Scholar
  6. Cockett MI, Bebbington CR and Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8: 662–667.Google Scholar
  7. Fiers W, Coutreras R, Haegemann G, Rogiers R, van de Voorde A, van Heuverswyn H, van Herreweghe J, Volckaert G and Ysebaert M (1978) Complete nucleotide sequence of SV40 DNA. Nature 273: 113–120.Google Scholar
  8. Haber DA, Beverley SM, Kiely ML and Schimke RT (1981) Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem 256: 9501–9510.Google Scholar
  9. Ichikawa D, Hashimoto N, Hoshima M, Yamaguchi T, Sawai K, Nakamura Y, Takahashi T, Abe T and Inazawa J (1996) Analysis of numerical aberrations of specific chromosomes by fluorescence in situ hybridization as a diagnostic tool in breast cancer. Cancer 77: 2064–2069.Google Scholar
  10. Kane SE, Troen BR, Gal S, Ueda K, Pastan I and Gottesman MM (1988) Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease. Mol Cell Biol 8: 3316–3321.Google Scholar
  11. Kaufman RJ and Sharp PA (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J Mol Biol 159: 601–621.Google Scholar
  12. Kaufman RJ, Sharp PA and Latt SA (1983) Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol 3: 699–711.Google Scholar
  13. 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 dihydrofolate reductase sequences in chinese hamster ovary cells. Mol Cell Biol 5: 1750–1759.Google Scholar
  14. Kaufman RJ (1993) Amplification and expression of transfected genes in mammalian cells. In: Kellems RE (ed) Gene Amplification in Mammalian Cells (pp. 315–343). Marcel Dekker Inc., New York.Google Scholar
  15. Kuriki H, Sonta S and Murata K (1993) Flow karyotyping analysis and sorting of the chinese hamster chromosomes: comparing the effects of the isolation buffers. J Clin Lab Anal 7: 119–122.Google Scholar
  16. Lee F, Yokota T, Otsuka T, Gemmell L, Larson N, Luh J, Arai K and Rennick (1985) Isolation of cDNA for a human granulocytemacrophage colony-stimulating factor by functional expression in mammalian cells. Proc Natl Acad Sci USA 82: 4360–4364.Google Scholar
  17. Mariani BD and Schimke RT (1984) Gene amplification in a single cell cycle in chinese hamster ovary cells. J Biol Chem 259: 1901–1910.Google Scholar
  18. Misawa S, Staal SP and Testa JR (1987) Amplification of the cmyc oncogene is associated with an abnormally banded region on chromosome 8 or double minute chromosomes in two HL-60 human leukemia sublines. Cancer Genet Cytogenet 28: 127–135.Google Scholar
  19. Morgan WF, Bodycot J, Fero ML, Hahn PJ, Kapp LN, Pantelias GE and Painter RB (1986) A cytogenetic investigation of DNA replication after hydroxyurea treatment: implications for gene amplification. Chromosoma 93: 191–196.Google Scholar
  20. Okayama H and Berg P (1983) A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol 3: 280–289.Google Scholar
  21. Okumura K (1992) Analysis of DNA in interphase nuclei by fluorescence in situ hybridization. Tanpakushitsu Kakusan Koso 37: 1546–1554.Google Scholar
  22. Omasa T, Higashiyama K, Shioya S, Suga K (1992) Effects of lactate concentrations on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng 39: 556–564.Google Scholar
  23. Omasa T, Itami S, Kameoka D, Katakura Y and Suga K (1996) Selection and stability for recombinant CHO cell line expressing human GM-CSF in gene amplification. Nagai K and Wachi M (eds), Animal Cell Technology: Basic and Applied Aspects (pp. 29–33). Kluwer Academic Publishers, The Netherlands.Google Scholar
  24. Pallavicini MG, DeTeresa PS and Wurm FM (1989) Use of fluorescence in situ hybridization to detect and monitor transfected and amplified sequences in recombinant CHO cells. Dev Biol Stand 70: 165–172.Google Scholar
  25. Pallavicini MG, DeTeresa PS, Rosette C, Gray JW and Wurm FM (1990) Effects of methotrexate on transfected DNA stability in mammalian cells. Mol Cell Biol 10: 401–404.Google Scholar
  26. Pinkel D, Straume T and Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934–2938.Google Scholar
  27. Potter H, Weir L and Leder P (1984) Enhancer-dependent expression of human κ immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 81: 7161–7165.Google Scholar
  28. Qumsiyen MB, Goorha S and Suttle DP (1993) Gene amplification and chromosome rearrangements: A study of a single cell lineage selected for amplification and deamplification of the UMP synthase gene. Cytogenet Cell Genet 62: 162–168.Google Scholar
  29. Rath H, Tlsty T and Schimke RT (1984) Rapid emergence of methotrexate resistance in cultured mouse cells. Cancer Res 44: 3303–3306.Google Scholar
  30. Reddy VB, Thimmappaya B, Dhar R, Subramanian KN, Zain BS, Pan J, Ghosh PK, Celma ML and Weissman SM (1978) The genome of simian virus 40. Science 200: 494–502.Google Scholar
  31. Ruiz JC and Wahl GM (1990) Chromosomal destabilization during gene amplification. Mol Cell Biol 10: 3056–3066.Google Scholar
  32. Selig S, Okumura K, Ward DC and Cedar H (1992) Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J 11: 1217–1225.Google Scholar
  33. Smith KA, Gorman PA, Stark MB, Groves RP and Stark GR (1990) Distinctive chromosomal structures are formed very early in the amplification of CAD genes in syrian hamster cells. Cell 63: 1219–1227.Google Scholar
  34. Southern PJ and Berg P (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1: 327–341.Google Scholar
  35. Stark GR (1993) Regulation and mechanisms of mammalian gene amplification. Adv Cancer Res 61: 87–113.Google Scholar
  36. Subramani S, Mulligan R and Berg P (1981) Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol 1: 854–864.Google Scholar
  37. Von Hoff DD (1991) New mechanisms of gene amplification in drug resistance (the episome model). In: Robbert F. Ozols (ed) Molecular and Clinical Advances in Anticancer Drug Resistance.Cancer Treatment and Research Vol. 57 (pp. 1–11). Kluwer Academic Publishers, The Netherlands.Google Scholar
  38. Weidle UH, Buckel P and Wienberg J (1988) Amplified expression constructs for human tissue-type plasminogen activator in chinese hamster ovary cells: instability in the absence of selective pressure. Gene 66: 193–203.Google Scholar
  39. Windle BE and Wahl GM (1992) Molecular dissection of mammalian gene amplification: new mechanistic insights revealed by analyses of very early events. Mutat Res 276: 199–224.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Tomohiro Yoshikawa
    • 1
  • Fumi Nakanishi
    • 1
  • Seima Itami
    • 1
  • Daisuke Kameoka
    • 1
  • Takeshi Omasa
    • 2
    Email author
  • Yoshio Katakura
    • 1
  • Michimasa Kishimoto
    • 1
  • Ken-ichi Suga
    • 1
  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversityOsakaJapan
  2. 2.Department of Biotechnology, Graduate School of EngineeringOsaka UniversityOsakaJapan

Personalised recommendations