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

将外源基因引入培养的哺乳动物细胞系中，增强其表达的技术不仅在分析基因功能方面具有重要意义，而且在重组产品的工业生产中也具有重要意义。在后一种情况下，有必要在稳定的转化子中增强引入基因的表达。用大肠杆菌β -半乳糖苷酶（β -gal）基因转染小鼠骨髓瘤细胞系P3/NS1-Ag4-1（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-1Z/gpt和CHO-Z/neo。与5-20 μ M 5-氮杂胞苷（5-azaC）孵育后，β - gal的比活性和总活性分别提高了2 ~ 3倍和1.5 ~ 2倍。当这些细胞系与5-10 mM 2-氨基嘌呤（2- ap）孵育12-24 h时，β - gal的活性也提高了2- 4倍。在这两种情况下，免疫滴定实验证实了β -Gal活性的增强是由于酶蛋白的增加。Northern blot分析显示，这些药物增强了β -Gal mRNA的表达。另一方面，2-AP不显著影响内源性基因如乳酸脱氢酶或β -肌动蛋白的表达。我们的结果表明，这些都是有用的药物，以上调引入基因的表达。

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.