Cytotechnology

, Volume 18, Issue 3, pp 167–172 | Cite as

Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter

  • Mitsuo Satoh
  • Hiromasa Miyaji
  • Tatsunari Nishi
  • Tamio Mizukami
  • Seiji Sato
  • Seiga Itoh
  • Mamoru Hasegawa
Article
  • 23 Downloads

Abstract

We have compared the level of expression of several enhancer/promoters in human lymphoblastoid Namalwa KJM-1 cells when fused to a common reporter gene. A cassette containing the pro-urokinase (pro-UK) coding sequence followed by the rabbit β-globin and simian virus 40 (SV40) 3′ nontranslated region was used for evaluation of the enhancer activity. Cells containing Moloney murine leukemia virus (Mo-MuLV) promoter had an average of 10–20 fold higher expression levels of pro-UK than those containing other promoters, such as SV40 early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Rous sarcoma virus (RSV) promoter and chicken β-actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 μg/106 cells/day and was constant for more than 6 months. Furthermore, the production of a high producer clone, obtained by using dhfr gene coamplification, reached 30–40 μg/106 cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.

Key words

efficient gene expression Moloney retroviral promoter Namalwa KJM-1 pro-urokinase 

Abbreviations

dhfr

dihydrofolate reductase

G-CSF

granulocyte colony-stimulating factor

hCMV

human cytomegalovirus

LTR

long terminal repeat

Mo-MuLV

Moloney murine leukemia virus

MTX

methotrexate

pro-UK

pro-urokinase

RSV

Rous sarcoma virus

SV40

simian virus 40

T3

triiodo-thyronine

TRE

thyroid-hormone responsive element

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References

  1. Foecking MK and Hofstetter H (1986) Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45: 101–105.Google Scholar
  2. Fregien N and Davidson N (1986) Activating elements in the promoter region of the chicken β-actin gene. Gene 48: 1–11.Google Scholar
  3. Gorman CM, Merlino GT, Willingham MC, Pastan I and Howard BH (1982) The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl. Acad. Sci. USA 79: 6777–6781.Google Scholar
  4. Granelli-Piperno A and Reich EJ (1978) A study of proteases and protease-inhibitor complexes in biological fluids. J. Exp. Med. 148: 223–234.Google Scholar
  5. Hosoi S, Miyaji H, Satoh M, Kurimoto T, Mihara A, Fujiyoshi N, Itoh S and Sato S (1991a) Optimization of cell culture consitions for production of biologically active proteins. Cytotechnology 5: S17–34.Google Scholar
  6. Hosoi S, Murosumi K, Sasaki K, Satoh M, Miyaji H, Hasegawa M, Itoh S, Tamaoki T and Sato S (1991b) Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered Namalwa KJM-1 cells. Cytotechnology 7: 25–32.Google Scholar
  7. Ishimoto A, Adachi A, Sakai K and Matsuyama M (1985) Long terminal repeat of Friend-MCF virus contains the sequence responsible for erythroid leukemia. Virology 141: 30–42.Google Scholar
  8. Laimins LA, Khoury G, Gorman C, Howard B and Gruss P (1982) Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus. proc. Natl. Acad. Sci. USA 79: 6453–6457.Google Scholar
  9. Miyaji H, Mizukami T, Hosoi S, Sato S, Fujiyoshi N and Itoh S (1990a) Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium. Cytotechnology 3: 133–140.Google Scholar
  10. Miyaji H, Harada N, Mizukami T, Sato S, Fujiyoshi N and Itoh S (1990b) Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium. Cytotechnology 4: 39–43.Google Scholar
  11. Miyaji H, Harada N, Mizukami T, Sato S, Fujiyoshi N and Itoh S (1990c) Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by dhfr gene coamplification method. Cytotechnology 4: 173–180.Google Scholar
  12. Mizukami T and Itoh S (1987) A new SV40-based vector developed for cDNA expression in animal cells. J. Biochem. 101: 1307–1310.Google Scholar
  13. Neuhaus G, Neuhaus-Url G, Gruss P and Schweiger H-G (1984) Enhancer-controlled expression of the simian virus 40 T-antigen in the green algaAcetabularia EMBO J 3: 2169–2172.Google Scholar
  14. Nishi T, Saito A, Oka T, Itoh S, Takaoka C and Taniguchi T (1984) Construction of plasmid expression vectors carrying theEscherichia coli tryptophan promoter. Agric. Biol. Chem. 48: 669–675.Google Scholar
  15. Sap J, Munoz A, Schmitt J, Stunnenberg H and Vennstrom B (1989) Repression of trabscription mediated at a thyroid hormone response element by the v-erb-A oncogene product. Nature 340: 242–244.Google Scholar
  16. Sato S, Kawamura K and Fujiyoshi N (1983) Animal cell cultuvation for production of biological substances with a novel perfusion culture apparatus. J. Tiss. Cult. Meth. 8: 167–171.Google Scholar
  17. Satoh M, Hosoi S, Miyaji H, Itoh S and Sato S (1993) Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability. Cytotechnology 13: 79–88.Google Scholar
  18. Speck NA and Baltimore D (1987) Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney leukemia virus enhancer. Mol. Cell. Biol. 7: 1101–1110.Google Scholar
  19. Thiesem H-J, Bosze Z, Henry L and Charnay P (1988) A DNA element responsible for the different tissue specificities of Friend and Moloney retroviral enhancers. J. Virol. 62: 614–618.Google Scholar
  20. Villiers JD, Olson L, Tyndall C and Schaffner W (1982) Transcriptional ‘enhancers’ from SV40 and polyoma virus show a cell type preference. Nucl. Acid. Res. 10: 7965–7976.Google Scholar
  21. Wasylyk B (1988) Enhancers and transcription factors in the control of gene expression. Biochim. Biophys. Acta 951: 17–35.Google Scholar
  22. Yanagi H, Yoshima T, Ogawa I and Okamoto M (1989) Recombinant human erythropoietin produced by Namalwa cells. DNA 8: 419–427. 3ptGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Mitsuo Satoh
    • 1
  • Hiromasa Miyaji
    • 1
  • Tatsunari Nishi
    • 1
  • Tamio Mizukami
    • 1
  • Seiji Sato
    • 1
  • Seiga Itoh
    • 1
  • Mamoru Hasegawa
    • 1
  1. 1.Tokyo Research LaboratoriesKyowa Hakko Kogyo Co.Machida-shi, TokyoJapan

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