Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 doi:10.1093/nar/gni192 Iterative in vivo assembly of large and complex transgenes by combining the activities of fC31 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 Received September 21, 2005; Revised October 17, 2005; Accepted November 25, 2005 ABSTRACT We have used the fC31 integrase to introduce large DNA sequences into a vertebrate genome and meas- ure 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 prob- lems 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 fC31 integrase and Cre recombinase to enable the iterative and serial integration of trans- genic 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 trans- genic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria. INTRODUCTION Site-speciﬁc recombinases are used to manipulate chromo- somal DNA sequence organization in vivo, in vitro (1) and in a range of different experimental systems. The enzymes Cre and Flp (2) are the most widely employed, both are members of the tyrosine recombinase family and catalyse reversible reactions between identical sites of 35 bp in the absence of any accessory proteins. The reversibility of the reactions catalysed by these enzymes has limited their applicability. They are generally used to promote deletion reactions since integration reactions are kinetically unfavourable. However Cre and Flp have also been used to engineer integrations and translocations in cells in culture where it has been possible to provide the enzyme transiently and trap the potentially unstable product, often but not always, by selection (3,4). A technology that would allow the construction of large segments of transgenic DNA would be of widespread utility. Thus it would be valuable to be able to assemble large tracts of transgenic DNA at deﬁned chromosomal loci in order to con- struct animal models of human genetic disease and to study the factors controlling expression of large genes. This goal has become more attractive with the ability to precisely and efﬁ- ciently manipulate DNA cloned in Escherichia coli by homo- logous recombination (5,6) and with the availability of a complete genome sequence and well-characterized libraries of bacterial artiﬁcial chromosome (BAC) clones. Similarly it would be useful to be able to build plants with multiple transgenes at a single locus. Finally it would be interesting to be able to construct synthetic bacterial genomes in order to identify the minimal set of genes consistent with independent life (7). Strategies describing how Cre or Flp might be used to promote irreversible integrations have been presented (8–10) However all of these approaches suffer from the limitation that they only allow one cycle of site-speciﬁc recombination and thus cannot be used iteratively. It is difﬁcult therefore to see how they could be used to construct large tracts of trans- genic DNA. *To whom correspondence should be addressed. Tel: +44 115 849 3244; Fax: +44 115 970 9906; Email: William.brown@nottingham.ac.uk Present address: Margaret C. M. Smith, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK 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@oxfordjournals.org