e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 PAGE 8 OF 14 Figure 5. A schematic illustration of how ISSI might occur. Details are discussed in the text. Step 1: A desired segment of DNA is cloned in what is here termed vector 1. Vector 1 consists of a circular piece of recom- binant DNA containing a second selectable marker gene and is abutted at the 50 end of the coding region by a loxP site. Two copies of an att site reciprocal to that already integrated into the genome ﬂank the insert cloning site in vector 1. In this example the att site would be an attB site. The recombinant DNA molecule in vector 1 is introduced into the cells con- taining the chromosome modiﬁed as described above and expressing both the Cre recombinase and unidirectional site-speciﬁc recombinase. Selection is applied such that cells expressing the marker gene in vector 1 survive. These cells would be expected to be enriched in those in which the Cre recombinase has catalysed recombination between the resident chromosomal loxP site and the loxP site cloned in vector 1. Step 2: As a result of the action of the Cre recombinase the insert contained within vector 1 is introduced into the chro- mosomal site and the two attB sites originally present in vector 1 are separated by the insert such that one is closer to the previously resident attP site. One of the two loxP products of the Cre mediated reaction is between the promoter and the marker gene contained within vector 1 while the second is between attP and attB sites. The next reaction to consider is that promoted by the unidirectional site-speciﬁc integrase which leads to the excision of the second loxP. The excision reaction may occur between the attP site and either of the two attB sites present in the incoming vector 1. If, as illustrated, the attB site nearest to the attP site participates in the unidirec- tional recombination reaction then the original marker gene and the loxP site adjacent to marker 1 is excised on a small circular fragment which would be expected to be lost from the nucleus at cell divison. The insert and attB site from vector 1 remain but loss of one of the two loxP sites renders the Cre catalysed integration irreversible. Alternatively if the attB site nearest to the second marker gene participates in the unidir- ectional recombination reaction then the ﬁrst marker, a loxP site and the insert in the ﬁrst vector will be excised. The ﬁrst of these two alternative unidirectional recombination reactions is necessary for the next step in the sequence because the integ- rated product can function as a substrate for another sequence of site-speciﬁc recombination reactions that can lead to the integration of a second insert. Step 3: A desired segment of DNA is cloned in what is here termed vector 2. Vector 2 consists of a circular piece of recom- binant DNA containing the original selectable marker gene and is abutted at the 50 end of the coding region by a loxP site. Two copies of an att site that can function as a substrate for the integrase and reciprocal to the site now integrated into the genome ﬂank the insert cloning site in vector 2. In this example the att site would be an attP site. The recombinant DNA molecule in vector 2 is introduced into the cells con- taining the chromosome modiﬁed as described above as a products of steps 1 and 2 and expressing the Cre recombinase and unidirectional site-speciﬁc recombinase. Selection is applied such that cells expressing the marker gene in vector 2 survive. These cells would be expected to be enriched in those in which the Cre recombinase has catalysed recombina- tion between the resident chromosomal loxP site and the loxP site cloned in vector 2. Step 4: As a result of the action of the Cre recombinase the insert contained within vector 2 is introduced into the chro- mosomal site and the two attP sites originally present in vector 2 are separated by the insert two such that one is closer to the attB site arising from the preferred reaction occurring in step 2 of the process. As above one of the two loxP products of this