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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10, 1788–95.
Sasaki, Y., Sone, T., Yoshida, S., Yahata, K., Hotta, J., Chesnut, J. D., Honda, T., and Imamoto, F. (2004) Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system. J Biotechnol 107, 233–43.
Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., and Brasch, M. A. (2004) Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res 14, 2111–20.
Sone, T., Yahata, K., Sasaki, Y., Hotta, J., Kishine, H., Chesnut, J. D., and Imamoto, F. (2008) Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning. J Biotechnol 136, 113–21.
Sasaki, Y., Sone, T., Yahata, K., Kishine, H., Hotta, J., Chesnut, J. D., Honda, T., and Imamoto, F. (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 promoter. J Biotechnol 136, 103–12.
Yahata, K., Maeshima, K., Sone, T., Ando, T., Okabe, M., Imamoto, N., and Imamoto, F. (2007) cHS4 insulator-mediated alleviation of promoter interference during cell-based expression of tandemly associated transgenes. J Mol Biol 374, 580–90.
Sone, T., Nishiumi, F., Yahata, K., Sasaki, Y., Kishine, H., Andoh, T., Inoue, K., Thyagarajan, B., Chesnut, J. D., and Imamoto, F. (2009) 21. Cell engineering using integrase and recombinase systems. in Emerging Technology Platforms for Stem Cells (Lakshmipathy, U., Chesnut, J. D., and Thyagarajan, B., Eds.) pp 379–394, John Wiley & Sons, Hoboken.
Messing, J. (1991) Cloning in M13 phage or how to use biology at its best. Gene 100, 3–12.
O’Gorman, S., Fox, D. T., and Wahl, G. M. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251, 1351–1355.
Chalberg, T. W., Portlock, J. L., Olivares, E. C., Thyagarajan, B., Kirby, P. J., Hillman, R. T., Hoelters, J., and Calos, M. P. (2006) Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol 357, 28–48.
Thyagarajan, B., Liu, Y., Shin, S., Lakshmipathy, U., Scheyhing, K., Xue, H., Ellerstrom, C., Strehl, R., Hyllner, J., Rao, M. S., and Chesnut, J. D. (2008) Creation of engineered human embryonic stem cell lines using phiC31 integrase. Stem Cells 26, 119–26.
Olivares, E. C., Hollis, R. P., and Calos, M. P. (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278, 167–76.
Kagawa, N., Kemmochi, K., and Tanaka, S. (2004) One-step adapter PCR method for HTP Gateway technology cloning. QUEST 1, 37–39.
Acknowledgments
The authors are grateful to Dr. Jonathan D. Chesnut (Lifetechnologies Corp.) for providing att signal sequences of MultiSite Gateway and information for construction.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sone, T., Imamoto, F. (2012). Methods for Constructing Clones for Protein Expression in Mammalian Cells. In: Hartley, J. (eds) Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 801. Humana Press. https://doi.org/10.1007/978-1-61779-352-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-352-3_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-351-6
Online ISBN: 978-1-61779-352-3
eBook Packages: Springer Protocols