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

Paper ID Volume ID Publish Year Pages File Format Full-Text
24530 43524 2008 9 PDF Available
Title
Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning
Abstract

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 Destination” 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 CmR 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.

Keywords
Multi-site gateway cloning; Multi-gene expression clone; High-throughput DNA cloning; Multiple recombination signals; Multiple DNA-fusion structure
First Page Preview
Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 136, Issues 3–4, 10 September 2008, Pages 113–121
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering