Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues

L. E. Rajagopalan, J. K. Burkholder, J. Turner, J. Culp, N. S. Yang, J. S. Malter

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

To increase transgenic production of granulocyte-macrophage colony- stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM- CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20-fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM- AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t( 1/2 ) = 95 minutes) than hGM-AUUUA mRNA (t( 1/2 ) = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.

Original languageEnglish (US)
Pages (from-to)2551-2558
Number of pages8
JournalBlood
Volume86
Issue number7
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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    Rajagopalan, L. E., Burkholder, J. K., Turner, J., Culp, J., Yang, N. S., & Malter, J. S. (1995). Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues. Blood, 86(7), 2551-2558.