PAGE 9 OF 14 Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 Cre mediated reaction is between the promoter and the marker gene contained within vector 1 while the second is between the attB site and attP site. Once again we now consider the reac- tion promoted by the unidirectional site-speciﬁc integrase which as in step 2 leads to the excision of the second loxP site. The excision reaction may occur between the attB site and either of the two attP sites originally present in the incoming vector 2. If, as illustrated, the attP site nearest to the attB site participates in the unidirectional recombination reaction then the marker gene 2 and the adjacent loxP site are excised to generate an unstable product. The insert and attP site from vector 2 remain but the loss from the cell of the small circular molecule generated by the integrase would render the Cre catalysed reaction irreversible. Were the attP site nearest to the second marker gene to participate in the unidirectional recombination reaction then marker 2, a loxP site and the second insert would be excised. The ﬁrst of these two altern- ative unidirectional recombination reactions is, of course, pre- ferred because as before the integrated product can function as a substrate for another sequence of site-speciﬁc recombination reactions that can lead to the integration of a third and sub- sequent inserts because it creates a target identical to that with which the process was started. The reaction series of steps 1–4 may therefore be repeated an arbitrary number of times. We refer to this strategy as ISSI. We have described the ISSI strategy as though the Cre promoted reaction occurred ﬁrst because this description is particularly easy to follow mechanistically. In fact the order of the two site-speciﬁc recombination reaction makes no dif- ference to the outcome and indeed they may occur concur- rently. The only feature of the reaction that is critical is which of the two attachment (att) sites in the incoming vector recom- bines with the attB or attP site adjacent to the resident select- able marker gene. Implementation of iterative site-specific integration We have implemented ISSI using a combination of the Cre recombinase and fC31 integrase. First of all we built the necessary plasmids, BAC and PAC vectors (Figure 6). In Figure 6. Plasmid and large insert vectors used in ISSI. (A) Plasmid attPloxPHytkattP used in the ISSI sequence shown in Figure 7. (B) Plasmid attBloxPBSRattBmod used in the ISSI sequence shown in Figure 7. (C) BAC vector fC31BAC attPCCAGloxPHyTkattP. This vector differs from the others in that it includes a promoter upstream of the selectable marker. We originally expected that this modification would improve the efficiency of the integration compared with a promoterless vector however comparisons revealed no difference in the respective efficiencies. This vector was used in the experiments shown in Figure 8. Promoterless BAC and PAC equivalents have been engineered (Supplementary Data). (D) PAC vector fC31PAC attBloxPBSRattBmod. This vector was used as the vector for the human X chromosome alphoid DNA in the experiment shown in Figure 8. A BAC equivalent has been engineered (Supplementary Data).