Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter
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- 作者：Yukari Sasaki ; Takefumi Sone ; Kazuhide Yahata ; Hiroe Kishine ; Junko Hotta ; Jonathan D. Chesnut ; Takeshi Honda ; Fumio Imamoto
- 关键词：Multisite Gateway cloning ; Multi-gene Expression clone ; Simultaneous introduction of multiple cDNAs ; Eukaryotic operon ; Controllable expression of transgenes
- 刊名：Journal of Biotechnology
- 出版年：2008
- 出版时间：10 September 2008
- 年：2008
- 卷：136
- 期：3-4
- 页码：103-112
- 全文大小：1250 K
文摘
Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.