Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning

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• 作者：Takefumi Sone ; Kazuhide Yahata ; Yukari Sasaki ; Junko Hotta ; Hiroe Kishine ; Jonathan D. Chesnut ; Fumio Imamoto
• 关键词：Multi-site gateway cloning ; Multi-gene expression clone ; High-throughput DNA cloning ; Multiple recombination signals ; Multiple DNA-fusion structure
• 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：113-121
• 全文大小：1022 K

文摘

Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a “Modular Destinationȁd; vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm^R and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.