Nucleic Acids Research, 2002, Vol. 30, No. 21 e115
© 2002 Oxford University Press
Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases
1 Division of Molecular Genetics and 2 Division of Pathology, Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, UK and 3 Max-Planck-Institute of Neurobiology, Am Klopferspitz 18a, 82152 Martinsried, Germany
*To whom correspondence should be addressed at Division of Molecular Genetics, Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, UK. Tel: +44 20 7608 6891; Fax: +44 20 7608 6863; Email: m.lauth{at}ucl.ac.uk
Work of the last decade has proven the ‘one gene– one product–one function’ hypothesis an oversimplification. To further unravel the emerging ‘one gene–multiple products–even more functions’ concept, new methods (such as subtle knock-in and tightly regulated conditional mutations) for the analysis of gene function in health and disease are required. Another class of improvements (such as tetraploid fusion and cassette exchange) addresses the efficiency with which targeted mutant strains can be generated. Recombinase-mediated cassette exchange (RMCE), which in theory is well suited for the rapid generation of multiple alleles of a given locus, is hampered by its low efficiency in the absence of selection and, especially in vivo, by the promiscuity of the participating recombinase recognition sites. Here we present a novel approach which circumvents this problem by the use of two independent recombinase systems. The strategy, which uses loxP on one and FRT on the other side of the cassette together with a Cre/Flpe expression vector, prevents excisive events and results in higher rates of cassette integration without selection than previously described. This method has a huge potential for the generation of allelic series in embryonic stem cells and, importantly, in pre-implantation embryos in vivo.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. Li, A. Xing, B. P. Moon, R. P. McCardell, K. Mills, and S. C. Falco Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange Plant Physiology, November 1, 2009; 151(3): 1087 - 1095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Louwerse, M. C.M. van Lier, D. M. van der Steen, C. M.T. de Vlaam, P. J.J. Hooykaas, and A. C. Vergunst Stable Recombinase-Mediated Cassette Exchange in Arabidopsis Using Agrobacterium tumefaciens Plant Physiology, December 1, 2007; 145(4): 1282 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pscherer, J. Schliwka, K. Wildenberger, A. Mincheva, C. Schwaenen, H. Dohner, S. Stilgenbauer, and P. Lichter Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma FASEB J, June 1, 2006; 20(8): 1188 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Dafhnis-Calas, Z. Xu, S. Haines, S. K. Malla, M. C. M. Smith, and W. R. A. Brown Iterative in vivo assembly of large and complex transgenes by combining the activities of {varphi}C31 integrase and Cre recombinase Nucleic Acids Res., December 15, 2005; 33(22): e189 - e189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Seibler, B. Kuter-Luks, H. Kern, S. Streu, L. Plum, J. Mauer, R. Kuhn, J. C. Bruning, and F. Schwenk Single copy shRNA configuration for ubiquitous gene knockdown in mice Nucleic Acids Res., April 14, 2005; 33(7): e67 - e67. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Osipovich, A. Singh, and H. E. Ruley Post-entrapment genome engineering: First exon size does not affect the expression of fusion transcripts generated by gene entrapment Genome Res., March 1, 2005; 15(3): 428 - 435. [Abstract] [Full Text] [PDF]
|
|