Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K
Institute for Medical Genetics, Kumamoto University Medical School, Japan.
Gene. 1989 Jul 15;79(2):269-77. doi: 10.1016/0378-1119(89)90209-6.
We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.
我们检测了位于鸡β-肌动蛋白基因5'侧翼区与第二外显子近端区域之间的1.3 kb序列的启动子活性。通过在小鼠L细胞中瞬时表达lacZ基因检测发现,该启动子区域显示出比猿猴病毒40(SV40)早期启动子或劳氏肉瘤病毒(RSV)长末端重复序列(LTR)更高的启动子活性。此外,用源自兔β-珠蛋白基因的3'剪接序列替换该启动子中的相应序列,导致β-半乳糖苷酶(βGal)的合成增加约2.5倍。将SV40 DNA复制起点(ori)引入携带这种杂交启动子(我们称之为AG启动子)的载体中,在产生SV40 T抗原的细胞BMT10中显著增强了βGal的产生。我们构建了一种有用的载体,其包含强AG启动子、多个独特的限制性酶切位点、一个SV40聚腺苷酸化信号和SV40 ori,用于在BMT10或COS细胞中瞬时表达cDNA。我们展示了该载体在BMT10细胞中高效产生白细胞介素-5的应用。