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Gene Gun-Mediated Human Erythropoietin Gene Expression in Primary Cultured Oviduct Cells from Laying Hens |
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H. Ochiai, H. M. Park, R. Sasaki, J. Okumura, T. Muramatsu |
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| Abstract |
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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 關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 60 kgf/cm2. The results showed that the erythropoietin mRNA concentration was significantly higher at 60 than 20 kgf/cm2. In experiment 2, the effects of supplementing the medium with fetal calf serum at 10%, and raising the shooting pressure from 60 to 80 kgf/cm2 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 10쨀 cells was not affected. Raising the shooting pressure from 60 to 80 kgf/cm2 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 60 kgf/cm2 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 |
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