Skip Navigation

Nucleic Acids Research 2005 33(22):e189; doi:10.1093/nar/gni192
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (2001K) Freely available
Right arrow Screen PDF (645K) Freely available
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Dafhnis-Calas, F.
Right arrow Articles by Brown, W. R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dafhnis-Calas, F.
Right arrow Articles by Brown, W. R. A.
Related Collections
Right arrow DNA transfer
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 15 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Iterative in vivo assembly of large and complex transgenes by combining the activities of {varphi}C31 integrase and Cre recombinase

Felix Dafhnis-Calas, Zhengyao Xu, Steve Haines, Sunir K. Malla, Margaret C. M. Smith and William R. A. Brown*

Institute of Genetics, Queen's Medical Centre, University of Nottingham Nottingham NG7 2UH, UK

*To whom correspondence should be addressed. Tel: +44 115 849 3244; Fax: +44 115 970 9906; Email: William.brown{at}nottingham.ac.uk

Received September 21, 2005. Revised October 17, 2005. Accepted November 25, 2005.

We have used the {varphi}C31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than ~150 kb we have established a method that we call; ‘Iterative Site Specific Integration’ (ISSI). ISSI combines the activities of {varphi}C31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.

Present address: Margaret C. M. Smith, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
P. A. Rowley, M. C. A. Smith, E. Younger, and M. C. M. Smith
A motif in the C-terminal domain of {phi}C31 integrase controls the directionality of recombination
Nucleic Acids Res., July 1, 2008; 36(12): 3879 - 3891.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Z. Xu, N. C. O. Lee, F. Dafhnis-Calas, S. Malla, M. C. M. Smith, and W. R. A. Brown
Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage {phi}BT1
Nucleic Acids Res., January 17, 2008; 36(1): e9 - e9.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
K. J. T. Venken and H. J. Bellen
Transgenesis upgrades for Drosophila melanogaster
Development, October 15, 2007; 134(20): 3571 - 3584.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. Ventura, C. Canchaya, A. Tauch, G. Chandra, G. F. Fitzgerald, K. F. Chater, and D. van Sinderen
Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum
Microbiol. Mol. Biol. Rev., September 1, 2007; 71(3): 495 - 548.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Gupta, R. Till, and M. C. M. Smith
Sequences in attB that affect the ability of {phi}C31 integrase to synapse and to activate DNA cleavage
Nucleic Acids Res., May 11, 2007; 35(10): 3407 - 3419.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Bischof, R. K. Maeda, M. Hediger, F. Karch, and K. Basler
An optimized transgenesis system for Drosophila using germ-line-specific {varphi}C31 integrases
PNAS, February 27, 2007; 104(9): 3312 - 3317.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.