Establishment of procedures to enhance the expression of introduced genes in mammalian cells.

建立增强哺乳动物细胞中导入基因表达的程序。

• 批准号: 04650877
• 负责人: OHMORI Hitoshi
• 金额: $ 1.34万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04650877/
• 关键词: mammalian cells; gene transfer; stable transfectant; 5-azacytidine; 2-aminopurine; beta-galactosidase; gene expression; recombinant product; 動物細胞株; 安定発現; mRNA; ノーザンブロット; タンパク合成

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.

提高导入哺乳动物细胞系的外源基因表达的技术不仅在基因功能分析中，而且在重组产物的工业生产中具有重要意义。我们用大肠杆菌的β-半乳糖苷酶（β-Gal）基因转染小鼠骨髓瘤细胞系P3/NS 1-Ag 4 -1（NS-1）和中国仓鼠卵巢细胞系CHO-K1，分离出稳定的转化子，分别命名为NS-1 Z/gpt和CHO-Z/neo。当这些细胞系与5-20 μ M的5-氮胞苷（5-azaC）孵育时，β-Gal的比活性和总活性分别提高2- 3倍和1.5- 2倍，当这些细胞系与5-10 mM的2-氨基嘌呤（2-AP）孵育12-24小时时，β-Gal的活性也提高2- 4倍。在这两种情况下，在免疫滴定实验中证实β-Gal活性的增强是由于酶蛋白的增加。北方印迹分析表明，这些试剂增加了β-Gal mRNA的表达，另一方面，2-AP对内源性基因如乳酸脱氢酶或β-actin的表达没有明显影响。

项目成果

Takahiro Tanigawa, Masaki Hikida, Toshiyuki Takai and Hitoshi Ohmori: "Enhancement of expression of introduced beta -galactosidase gene by 2-aminopurine in mammalian cell lines." J.Ferment.Bioeng.Vol.76. 145-147 (1993)

Takahiro Tanikawa、Masaki Hikida、Toshiyuki Takai 和 Hitoshi Ohmori：“在哺乳动物细胞系中通过 2-氨基嘌呤增强引入的 β-半乳糖苷酶基因的表达。”

Hitoshi Ohmori: "DNA transfection enhanced by heat" Rice Biotechnology Quartery. 11. 24- (1992)

Hitoshi Ohmori：“热增强 DNA 转染”水稻生物技术季刊。

Takahiro Tanogawa: "Enhancement of expression of introduced gene by 5-azacytidine in mammalian cell lines." J.Fermt.Bioeng.(1993)

Takahiro Tanokawa：“5-氮杂胞苷在哺乳动物细胞系中增强导入基因的表达。”

Takahiro Tanigawa: "Enhancement of expression of an introduced β-galactosidase gene by 2-aminopurine in mammalian cells." Journal of Fermentation and Bioengineering. 76. 145-147 (1993)

Takahiro Tanikawa：“通过 2-氨基嘌呤增强哺乳动物细胞中引入的 β-半乳糖苷酶基因的表达。”《发酵与生物工程杂志》76. 145-147 (1993)。

Toshiyuki Takai and Hitoshi Ohmori: "Enhancement of DNA transfection efficiency by heat treatment of cultured mammalian cells." Biochim.Biophys.Acta. Vol.1129. 161-165 (1992)

Toshiyuki Takai 和 Hitoshi Ohmori：“通过热处理培养的哺乳动物细胞提高 DNA 转染效率。”