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Gene Gun-Mediated Human Erythropoietin Gene Expression in Primary Cultured Oviduct Cells from Laying Hens

  • Ochiai, H. (Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University) ;
  • Park, H.M. (Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University) ;
  • Sasaki, R. (Department of Food Science and Technology, Faculty of Agriculture, Kyoto University) ;
  • Okumura, J. (Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University) ;
  • Muramatsu, T. (Department of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University)
  • Received : 1997.12.11
  • Accepted : 1998.06.08
  • Published : 1999.01.01

Abstract

Factors affecting gene gun-mediated expression of the human erythropoietin gene were investigated in primary cultured oviduct cells from laying hens. The human erythropoietin gene was transfected by a gene gun method at $1.25{\mu}g$ per dish, and cultured in a synthetic serum-free medium for 72 hrs. The concentration of human erythropoietin mRNA was determined by RNA : RNA solution hybridization. In experiment 1, the effect of changing the shooting pressure of DNA-coated microparticles with nitrogen gas was tested at 20 and $60kgf/cm^2$. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than $20kgf/cm^2$. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to $80kgf/cm^2$ on the cell number and erythropoietin gene expression were examined. Although supplementation with fetal calf serum significantly increased the cell numbes compared with no supplemented controls (p < 0.05), erythropoietin mRNA concentration per $10^3$ cells was not affected. Raising the shooting pressure from 60 to $80kgf/cm^2$ did not affect either of the parameters, In experiment 3, the effect of supplementing ascorbate 2-phosphate at 0.5 mM was tested. The results indicated that the ascorbate supplementation significantly increased the cell number (p < 0.05), and tended to increase erythropoietin mRNA concentration (p < 0.1). Thus, for human erythropoietin gene expression by using the gene gun method, shooting pressure with nitrogen gas should be sufficient at $60kgf/cm^2$ and supplementation with ascorbate phosphate would be useful to enhance not only the cell proliferation but also erythropoietin gene expression.

Keywords

Human Erythropoietin;Gene Gun;Primary Cultures;Oviduct Cells;Laying Hens