e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 order to do this we noted the results of others which demon- strated that fC31 attB sites are less recombinationally active when integrated into vertebrate genomic DNA than when pre- sent in cloned DNA (21). The fC31 attB site includes seven CpG sites that are potentially methylatable in vertebrate genomic DNA. This suggested to us that cytosine methylation at the 50 position within these CpG dinucleotides was inhib- iting the ability of a genomic attB site to participate in site- speciﬁc recombination reactions. We therefore investigated the effect of CpG methylation of the attB site upon its ability to participate in site-speciﬁc recombination reactions in vitro. These results (S. K. Malla unpublished data) demonstrated that CpG methylation does indeed inhibit the ability of the attB site to participate in fC31 mediated site-speciﬁc recombination in vitro. We therefore devised an unmethylatable analogue of the attB site termed attBmod and have shown that it functions as efﬁciently as the unmethylated native attB in vivo. We have therefore used attBmod in the ISSI plasmids shown in Figure 6. Although the details of the work describing the effects of CpG methylation of the attB site upon fC31 site-speciﬁc recomb- ination reaction are unpublished the sequence of attBmod is provided in the Materials and Methods. We also used bi- sulphite sequencing to investigate whether the attB site in the CCAG attB HyTk construct used in the previous experi- ments was methylated and ﬁnd that it was not (S.K. Malla unpublished data). The initial conclusions and the numbers in Table 1 are therefore not complicated by the consequences of attB site methylation in vivo. The attB site in the CCAG attB HyTk construct is located adjacent to the CpG island deﬁned by the chicken b-actin promoter within the CCAG promoter and thus would not be expected to be readily methylated in vivo. However in the work described by Belteki et al. (21) the PAGE 10 OF 14 attB site lies outside the transcribed region of the gene and not within a CpG island and it may therefore be readily methyl- ated. It thus remains to be established whether an inappropri- ately placed attB site can be methylated in vivo and when it is so methylated to what extent this inhibits site-speciﬁc recomb- ination. Experiments to establish this point are in progress (S. K. Malla and W. R. A. Brown). The genes that we used in the integrating plasmids were those conferring resistance to the antibiotics blasticidin (16) (BSR) and hygromycin (17) (HyTk). In order to implement ISSI we targeted the seed construct CCAG loxP HyTk attP (Figure 5A) to the 49B(A)A9 mini- chromosome in the same way as we had targeted the CCAG attB HyTk construct (Figure 1B). We then introduced sequen- tially the CCAG 50nls fC31 integrase IRES zeo (Figure 2A) and the CCAG Cre IRES ecogpt (Supplementary Data) plas- mids that direct expression of the fC31 integrase tagged at the N-terminus with a nuclear localization sequence and the Cre recombinase, respectively. This generated several cells lines which should be capable of mediating the ISSI sequence. We used one of these, termed ISSI 2, for our proof of principle experiments. In the ﬁrst of our proof of principle experiments we used plasmids to execute two complete cycles of this strategy (Figure 7A). This led to the integration of a concatamer of four pBlueScript and accompanying polylinker sequences into the target. The presence of four other plasmid vector DNA molecules integrated into background of this cell line pre- cluded direct analysis of the integrated DNA and so we used restriction enzyme digestion, ﬁlter hybridization and indirect end labelling to conﬁrm the predicted structures of the DNA integrated during the ISSI sequence. DNA extracted Figure 7. ISSI of plasmid vector molecules into mini-chromosome A9 CCAG loxP HyTk attP in DT40 cells. (A) Illustrates the iterations of plasmid integration reactions into the CCAG loxP HyTK attP site present in the starting cell line ISSI 2 and analysed in (B). The positions of the substrate and product attachment (att) sites and of the restriction enzyme sites used in the mapping are indicated. The cassettes used in these transfections were alternately attB loxP BSR attBmod and attP loxP HyTk attP. (B) Filter hybridization analysis of genomic DNA extracted from the cell lines generated in the sequence indicated in A using probes recognizing the selectable marker genes used in the ISSI sequence. The cross hybridization to chicken DNA seen with the HyTk probe at 25 kb serves as a loading control in this analysis.