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Cytotechnology

, Volume 37, Issue 3, pp 133–141 | Cite as

Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line

  • Yoshihisa Kuwana
  • Kikuko Funayama
  • Hiromasa Miyaji
  • Mamoru Hasegawa
  • Hajime Yoshida
  • Seiga Itoh
Article

Abstract

The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of ∼40 mg ml-1 in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the disruption of expression of the immunoglobulin heavy chain proteins with a frequency of 1 in 10 ∼ 100 G418-resistance transfectants. One of the monoclonal antibody-deficient transfectants produced25 ng ml-1 of granulocyte colony-stimulating factor in the supernatant of its cell culture the number of molecules of which corresponds to that of the monoclonal antibody originally produced by KM50. However, when this transfectant was injected intraperitoneally, it produced only a 9 μg ml-1 concentration of granulocyte colony-stimulating factor in ascites, which is approximately 3 orders of magnitude less than the monoclonal antibody. This method may be applicable to production of other recombinant proteins, although further optimization in the conditions of production would be needed in order to reach much higher yields.

Granulocyte colony-stimulating factor (G-CSF) Homologous recombination Immunoglobulin Production Promoter Recombinant proteins 

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Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Yoshihisa Kuwana
    • 1
  • Kikuko Funayama
    • 1
  • Hiromasa Miyaji
    • 1
  • Mamoru Hasegawa
    • 1
  • Hajime Yoshida
    • 1
  • Seiga Itoh
    • 1
  1. 1.Tokyo Research LaboratoriesKyowa Hakko Kogyo, Co. Ltd.TokyoJapan

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