Methods for Constructing Clones for Protein Expression in Mammalian Cells
- 4 September 2011
- book chapter
- Published by Springer Science and Business Media LLC
- Vol. 801, 227-250
- https://doi.org/10.1007/978-1-61779-352-3_15
Abstract
Multisite Gateway technology is a DNA cloning method based on in vitro site-specific recombination that is becoming increasingly popular because it allows quick and highly efficient assembly of multiple DNA fragments into a vector backbone. In the conventional Gateway Multisite strategy, cloning of multiple DNA fragments requires recombination of multiple entry clones with a single destination vector. The limitation of this approach is that as the number of entry clones increases, the efficiency of the assembly reactions decreases due to difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, we have devised methods to generate modular expression clones, modular entry clones, and modular destination vectors. These allow many DNA fragments to be assembled stepwise into complex expression clones. We describe here how to construct these intermediate clones and vectors, and how to use these modules to construct expression clones comprising ten or more DNA segments. These principles can be applied to make multicomponent DNAs for many applications.Keywords
This publication has 12 references indexed in Scilit:
- Cell Engineering Using Integrase and Recombinase SystemsPublished by Wiley ,2009
- Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloningJournal of Biotechnology, 2008
- 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 promoterJournal of Biotechnology, 2008
- cHS4 Insulator-mediated Alleviation of Promoter Interference during Cell-based Expression of Tandemly Associated TransgenesJournal of Molecular Biology, 2007
- Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 IntegraseThe International Journal of Cell Cloning, 2007
- Integration Specificity of Phage ϕC31 Integrase in the Human GenomeJournal of Molecular Biology, 2006
- Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression ClonesGenome Research, 2004
- Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway systemJournal of Biotechnology, 2004
- Cloning in M13 phage or how to use biology at its bestGene, 1991
- Recombinase-Mediated Gene Activation and Site-Specific Integration in Mammalian CellsScience, 1991