PAGE 7 OF 14 Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 Figure 4. fC31 integrase mediated integration of alphoid DNA inserts cloned in a BAC trapping vector into mini-chromosome A9 CCAG attB HyTk in DT40 cells. (A) Pulsed-field gel analysis of Y alphoid DNA sequences of 30, 70, 110 and 140 kb cloned in BAC vector AprafC31attPneo/pBAC3e. (B) Restriction and filter hybridization analysis of the mini-chromosome A9 CCAG attB HyTk. Following digestion with SacI the DNA was size fractionated by PFGE. Hybridization analysis was carried out using a probe specific for alphoid DNA. The arrows indicate the 90 and 15 kb alphoid DNA fragments endogenous to this starting mini-chromosome. The marker used in this gel was a collection of multimers of the phage l cI857 genome (48.5 kb). (C) Restriction and filter hybridization analysis of the mini- chromosome A9 CCAG attB HyTk following site-specific integration of the BACs containing inserts of Y chromosome alphoid DNA of the indicated sizes. Following digestion with SacI the DNA was size fractionated by PFGE. Hybridization analysis was carried out using a probe specific for alphoid DNA. The arrows indicate the 90 and 15 kb alphoid DNA fragments endogenous to the starting mini-chromosome. The left most track in each panel was of a SacI digest of DNA extracted from a site-specific recombinant between a BAC containing a 33 kb insert and the mini-chromosome A9 CCAG attB HyTk. The penultimate track in the 110 kb panel includes degraded DNA but was shown to derive from an undeleted integrant in a separate gel. The marker used in this gel was a collection of concatamers of the intact and XhoI digested phage l cI857 genome (48.5 kb). Sizes in Materials and Methods. (D) PCR analysis of the recombinant attL and attR sites generated by site-specific recombination between the incoming BAC DNA and the resident mini-chromosome A9 CCAG attB HyTk in a selection of clones containing re-arranged DNA. The aberrantly sized attL site in the type 2 re-arrangement indicated with an asterisk is consistent with deletion of sequences between the two primer binding sites. Iterative site-specific integration of transgenic DNA fragments The results described in Table 2 and Figure 4 demonstrate that while it is possible to integrate large DNA fragments clones containing the intact integrated molecule are a small percent- age of the stably transfected clones if the incoming molecule is much bigger than 100 kb. It would be useful however to be able to assemble larger stretches of transgenic DNA than are implied by this ﬁgure. For example it would be valuable to be able to build transgenic mice containing human gene families of medical importance. This goal could be achieved by com- bining the activities of a unidirectional and reversible recom- binase as outlined in Figure 5. In this strategy a modiﬁed selectable marker gene is ﬁrst introduced into a chromosome and then used as a seed from which the transgenic sequences are grown. The modiﬁcations of the selectable marker are envisaged as follows; a site for a reversible recombinase; e.g. a loxP site for Cre recombinase is placed between the promoter and coding region of the gene and an att site for a unidirectional site-speciﬁc recombinase such as fC31 integ- rase is placed at the 30 end of the gene. In this example the site is chosen, arbitrarily to be an attP site. Plasmids that direct expression of the Cre recombinase and a unidirectional site- speciﬁc recombinase are then introduced into the cells con- taining the modiﬁed selectable marker gene. This modiﬁed selectable marker gene can then be used as the site from which inserts in circular DNA molecules; typically cloned in plasmid, cosmid or BAC vectors are iteratively introduced into the chromosome as described in the following.