Multi-gene Gateway clone design for expression of multiple heterologous genes in living cells: Conditional gene expression at near physiological levels
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- 作者：Yahata ; Kazuhide ; Kishine ; Hiroe ; Sone ; Takefumi ; Sasaki ; Yukari ; Hotta ; Junko ; Chesnut ; Jonathan D. ; et. al.
- 关键词：Multisite Gateway cloning ; Multi-gene expression clone ; Simultaneous introduction of multiple cDNAs ; Low expression promoters ; Controllable expression of transgenes
- 刊名：Journal of Biotechnology
- 出版年：2005
- 出版时间：August 4, 2005
- 年：2005
- 卷：118
- 期：2
- 页码：123-134
- 全文大小：417 K
文摘
Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1α or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CPα1 and CPβ2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4 % of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed.