1993 Fiscal Year Final Research Report Summary
Establishment of procedures to enhance the expression of introduced genes in mammalian cells.
| Project/Area Number |
04650877
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | Okayama University |
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Principal Investigator |
OHMORI Hitoshi Professor Department of Biotechnology, Faculty of Engineering OKayama University, 工学部, 教授 (70116440)
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| Project Period (FY) |
1992 – 1993
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| Keywords | mammalian cells / gene transfer / stable transfectant / 5-azacytidine / 2-aminopurine / beta-galactosidase / gene expression / recombinant product |
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| Research Abstract |
Techniques that allow the enhancement of expression of exogenous genes introduced into cultured mammalian cell lines are of great importance not only in analyzing gene functions, but also in the industrial production of recombinant products. In the latter case, it is necessary to enhance expression of the introduced gene in stable transformants.
We transfected a mouse myeloma cell line, P3/NS1-Ag4-1(NS-1), and a Chinese hamster ovary cell line, CHO-K1 with the beta-galactosidase (beta-Gal) gene of Escherichia coil, and isolated stable transformants designated as NS-1Z/gpt and CHO-Z/neo, respectively. When these cells were incubated with 5-20 mu M 5-azacytidine (5-azaC), the specific and total activity of beta -Gal were enhanced 2- to 3-fold and 1.5- to 2-fold, respectively.
When these cell lines were incubated with 5-10 mM 2-aminopurine (2-AP) for 12-24 h, the activity of beta -Gal was also enhanced 2- to 4-fold. In both cases, it was confirmed in immunotitration experiments that the enhancement of beta -Gal activity was due to the increase of the enzyme protein. Northern blot analysis revealed that these agents augmented the expression of beta -Gal mRNA.On the other hand, 2-AP did not significantly affect the expression of an endogenous gene like lactate dehydrogenase or beta -actin.
Our results suggest that these are useful agents for up-regulating the expression of introduced genes.
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