PAGE 11 OF 14 from the cells was analysed by digestion with an enzyme that does not cut in pBluescript; NcoI, and then analysed (Figure 7B) either with a probe that recognizes the gene conferring resistance to the antibiotic Blasticidin (BSR) or with a probe from the hygromycin resistance gene present in the original target and in the products of the second and fourth plasmid integration reactions. These cell lines are referred to in Figure 7B by the number of the transfection from which they were derived which is indicated to the left of Figure 7A. The sizes and distribution of the cognate restric- tion fragments in these cells lines were precisely as predicted. At each step in the sequence of transfections illustrated in Figure 7 we characterized 10 individual clones in order to identify those in which the fC31 att site 50 to the incoming marker had recombined with the resident substrate site and which therefore had a structure that made them potentially available for a subsequent round of integration. At each step we found that all the recombination events were with one or other of the two sites and that each site had recombined with similar efﬁciency. It seemed to us possible that since ISSI requires the coordinated action of two site-speciﬁc recom- binases for a stable integration of the desired structure it would be less efﬁcient than the integration reaction promoted by the fC31 integrase alone. This was not so and ISSI appeared to be at least as efﬁcient as the single fC31 reaction alone for plasmid (Table 1) integrations. In the second proof of principle experiment we started with the product of transfection 267 described in the plasmid ISSI experiment and successively introduced a 70 kb alphoid DNA array derived from the human Y chromosome cloned in a CCAG loxP HyTk attP attP BAC and a 80 kb array of alphoid DNA derived from the human X chromosome cloned in a loxP BSR attB attB BAC (Figure 8A and B). This led to the isola- tion of cell line 293 containing the Y alphoid DNA and then to cell line 419 containing both the Y and X alphoid DNA (Figure 8B). We characterized these cell lines at successively higher resolution by three methods. Firstly we demonstrated that we could detect the acquisition of the alphoid DNA arrays by PFGE of the intact mini-chromosomes (Figure 8C). Integ- ration of the X chromosome alphoid DNA was indicated by speciﬁc hybridization and of both sequences by a slight increase in the size of the mini-chromosome. Secondly we used restriction enzyme digestion, PFGE and ﬁlter hybridiza- tion to establish physical linkage of the sequences. This ana- lysis made use of the sequence of the junctions and the low sequence complexity of the two sequences. Neither BglII nor BclI cut either alphoid DNA array but BglII cuts the polylinker junction between the two arrays. Consequently BglII releases each different array separately but BclI releases them together on a single large fragment of 170 kb in size and thus reveal their linkage (Figure 8D). The BclI fragments are 20 kb longer than the integrated DNA because the integration site itself lies on a 20 kb BclI fragment. The 15 kb alphoid DNA cognate fragment is increased in size in the mini-chromosomes in cell lines 293 and 419 as compared with the mini-chromosome in clone NLS2. This increase arises because of the introduction of the pBluescript molecule during transfection 267 immedi- ately prior to the generation of cell line 293 (Figure 8A). Finally we established the presence of the predicted products and substrates of the site-speciﬁc recombination reactions in the starting line ISSI 2 and in the three cell lines used in the Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 sequence. There are nine such junctions (Figure 8B). Their presence or absence in the cell lines is as predicted (Figure 8E); sequence analysis (data not shown) conﬁrms the identity of the product in each case. These results thus prove that the ISSI functions as envisaged. The efﬁciency of the ISSI BAC trans- formation reactions is greater (Table 2) than seen with the fC31 integrase alone which is consistent with the results seen with plasmids. DISCUSSION The experiments described here demonstrate that the fC31 integrase speciﬁcally and efﬁciently promotes site-speciﬁc integration in chicken DT40 cells. The observation that these proteins function in vertebrate cells is consistent with earlier work showing that the puriﬁed fC31 integrase pro- motes unidirectional exchange reactions between attB and attP sites in vitro (11) and with some aspects of the results of others who have studied either the fC31 and R4 (21,22) integrases in mammalian cells. We have shown that integration of intact 140 kb BACs into vertebrate cells is very inefﬁcient because they usually break before they integrate. The breakage does not appear to reﬂect the integrity prior to electroporation because the DNA was puriﬁed as closed circular by equilibrium centrifugation on a caesium chloride gradient in the presence of ethidium bromide and was assessed as intact by PFGE. Although there have been some reports of the successful electroporation of BACs into mammalian cells (23) others have reported problems (24) and have developed episomal vector systems in order to partially overcome them. We therefore conclude that the problem of integrating intact, large BACs into genomic DNA might be a general one. We have addressed the problem of integrating large seg- ments of DNA by establishing a strategy that combines the activities of the fC31 integrase and Cre recombinase to permit iterative integration of transgenic sequences. The strategy that we have implemented is termed ISSI is just one of several that one may envisage in which the activities of one or more uni- directional enzymes are combined with either the activity of a reversible recombinase or homologous recombination to per- mit serial or iterative integration. In one of these the activity of Cre is combined with that of two homing nucleases or mega- nucleases, oligonucleotides and T4 ligase and has been used for multiple rounds of integration into cloned DNA in vitro (25). The use of the two meganucleases rather than a single unidirectional enzyme allowed a single unambiguous resolu- tion product to be generated at each half of the cycle but this is not necessary when one wishes to assemble an array in vivo because one would wish to check the integrants for their integ- rity at each stage. Qualitatively ISSI works exactly as anticip- ated but quantitatively ISSI works better than one might have supposed. The reaction sequence that leads to the integration of one marker and excision of another is not known but the efﬁciency with which these steps occur relative to that of a single fC31 integrase integration reaction suggests that the fC31 integrase and the Cre are cooperating at the rate-limiting step. Such cooperativity could arise if the rate-limiting step was the association of the incoming DNA with the genomic target and both the Cre recombinase and the fC31 integrase bound to each of their respective target sites. This would allow