PAGE 13 OF 14 Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 for a bivalent association which would be expected to increase the afﬁnity of participating DNA molecules for one another and their overall rates of reaction. The observation that the two attachment sites in the incom- ing circular DNA recombine equally efﬁciently with the recip- rocal genomic attachment site is at ﬁrst surprising. A linear representation of a coordinate reaction process might lead one to suppose that the 30 site in the incoming DNA would be stereo-chemically favoured. Our observation may reﬂect a two-step process as envisaged in Figure 5 or the fact that both participating DNA molecules are folded in three dimen- sions and that recombination between the loxP sites favour neither the 30 nor the 50 attachment sites. Vertebrate centromeres are typically composed of several hundred kilobasepairs of tandemly repeated sequences. We have demonstrated that ISSI will enable us to build such struc- tures. We anticipate that ISSI may also be applied to other problems of experimental transgenesis where it is necessary to combine and assemble multiple transgenes. In some of these it will be simpler to ‘stack’ (25) the transgenes in vitro prior to their introduction into the genome but in others, where one wishes, for example, to introduce longer stretches of DNA, this will be impractical and in vivo assembly will be the only option. Examples include the assembly of large genomic regions for the purpose of investigating distally located control elements, reconstruction of complex loci such as the major histocompatibility complex as transgenes and the synthesis of bacterial genomes (7). Although we have carried out two proofs of principle experiments it remains to be proven how well ISSI works in cells other than those of the chicken DT40 line. We have argued in the introduction that our experi- mental approach is robust in so far as we have shown that we can integrate unstable sequences into a hyper-recombinogenic cell line. One limitation may be how well the unidirectional integrase works in different cell types. fC31 integrase itself has been shown to function in mouse, human, ﬁssion yeast and Drosophila cells [reviewed in (26)]. Some uncertainty also surrounds the ease with which large DNA fragments may be introduced into both eukaryotic and prokaryotic cells. Nev- ertheless BACs are routinely introduced into E.coli cells (27) and have been transferred into mouse and human ﬁbroblasts (24). Similarly large DNA fragments may be readily intro- duced into both budding (28) and ﬁssion yeast (29). This body of work suggests that size of the transfected DNA will not fundamentally limit the implementation of ISSI in a wide variety of cell types. Consistently in unpublished data we have used three rounds of ISSI to construct a 210 kb transgene in Chinese hamster ovary cells. In these experiments the fBT1 integrase (30) rather than the fC31 integrase was used as the unidirectional component in the reaction. Despite these argu- ments and encouraging preliminary results only sustained application in a variety of systems will establish the utility and limitations of our technique. SUPPLEMENTARY DATA Supplementary Data is available at NAR Online. ACKNOWLEDGEMENTS We thank David Brook for discussion and Alistair Chambers for comments on the manuscript. The work was supported by the EU (the DT40 cell line as a genetic model. Contract QLRT- 1999-00923), the Leverhulme Trust, the BBSRC and the University of Nottingham through a studentship to S.K.M. Funding to pay the Open Access publication charges for this article was provided by the JISC. Conflict of interest statement. None declared. REFERENCES 1. Sauer,B. 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