e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 Here we investigate the use of an integrase from the Strep- tomyces’ phage fC31 (11); this integrase belongs to the serine recombinases family of proteins and is thus releated to the resolvase/invertases. fC31 integrase is one of an expanding group of so-called ‘large serine recombinases’ because of their much higher molecular weight compared with the resolvase/ invertases. The serine recombinases differ evolutionarily and mechanistically from the tyrosine recombinases such as Cre and Flp. Several phage integrases of the large serine recom- binases have been shown to promote unidirectional or irre- versible recombination between non-identical sites of 50 bp in vitro in the absence of additional proteins (11,12). They would thus be potentially ideal for engineering precisely those types of re-arrangement that cannot be promoted by Cre and Flp. Several such phage integrases have been shown to be active in vertebrate cells (13). However these reports did not include unambiguous measurements of the efﬁciency of the reactions promoted by these proteins but emphasized the suggestion that exchange reactions could occur ectopically between one or other of the speciﬁc sites and several genomic sites (13). Such ectopic recombination would, of course, com- promise the utility of these enzymes for genome engineering but, it was argued, enhance their utility as reagents for gene therapy. Here we measure the efﬁciency of the class V serine recom- binase: fC31 integrase (11), in chicken DT40 cells. We show that this enzyme can be used to promote the efﬁcient integ- ration of plasmids and fragments as large as 100 kb into ver- tebrate chromosomes. Sequences larger than 100 kb are more frequently deleted than integrated intact. In order to overcome this limitation we have established a method that enables the iterative integration of transgenic DNA sequences by combin- ing the site-speciﬁc recombinase activities of the fC31 integ- rase and Cre recombinase. This method which we call ‘iterative site-speciﬁc integration’ (ISSI) potentially enables the reconstitution of transgenes of unlimited length and deﬁned sequence organization. We describe proof of principle experiments in which we have constructed an array of plasmid DNA sequences and a complex array of vertebrate centromeric sequences of 150 kb in size. Although our experiments have been carried out in vertebrate cells the principle that underlies ISSI is simple and applicable to transgenesis in all organisms: plants and bacteria as well as animals. ISSI should thus enable the reconstruction of complete genomes in bacteria and of biosynthetic pathways encoded by many different genes in transgenic organisms. A merit of assembling transgene arrays at a single locus in whole organisms using a technique such as ISSI is that doing so would avoid the difﬁculties that would arise as a result of the segregation of independent transgenic loci during meiosis. In our proof of principle experiments we have used site- speciﬁc recombination to introduce long, deﬁned tracts of tandemly repeated DNA into hyper-recombinogenic cells derived from the DT40 cell line. Thus we have demonstrated that long tracts of repeated DNA sequences can be introduced into a cell line where such sequences might be expected to be particularly unstable. These experiments therefore suggest that ISSI may be useful in a wide variety of cell types and for a variety of different sequences. However some cell types may be less readily transfected with large DNA than DT40 cells or may process such sequences differently from DT40 cells. PAGE 2 OF 14 Serine recombinases have been shown to work in many cells types but there may be some where they do not and in these implementation of ISSI would be impractical. MATERIALS AND METHODS Plasmid construction Plasmids were constructed by standard techniques including recombineering using the DY380 strain of Court and cowork- ers (14). The sequences of the plasmids and of the integrases used in this work can be obtained from http://www. nottingham.ac.uk/genetics/brown/genomeengineering.php. The Y chromosome alphoid (DYZ3) DNA BAC used in these experiments was 442H19 from the CITB Human BAC DNA (B and C libraries) Release 4. The original BAC had an insert of 150 kb of human Y alphoid DNA but was unstable in culture and derivatives of varying sizes could be isolated from col- onies left on plates for several weeks. The BAC 442H19 was a kind gift of Jonathan Flint and colleagues of the Wellcome Trust Centre for Human Genetics, Oxford. The X chromosome alphoid (DXZ1) DNA was derived from a PAC subclone of BAC RPCI-11 242E23 kindly provided by Hunt Willard and colleagues. The nuclear localization signal used to tag the fC31 was derived from the large T antigen of SV40 virus and included the residues MPKKKRKV. The attP and attB used sites were as follows: gtagtgccccaactggggtaaccttt- gagttctctcagttgggggcgtag and ccgcggtgcgggtgccagggcgtg- cccttgggctccccgggcgcgtactcc for the standard integration. The sequence of the modiﬁed attB site used in the ISSI con- structs was accgcggtgcgggtgccagggtgtgcccttgggctccccaggg- cacccctccac. In each case these att sites were engineered into plasmids using synthetic DNA provided by Invitrogen. The plasmids were checked by restriction site mapping and in all cases by sequencing across the att sites. The gene encoding resistance to apramycin was as described by Kuhstoss et al. (15). The blasticidin resistance (BSR) (16) gene was a kind gift from Hiroshi Arakawa of the GSF, Munich. The hygromycin resistance gene used was present in the counter selectable hygromycin–thymidine kinase fusion (HyTk) (17). The CCAG promoter (18) was a kind gift of Ian Chambers (Edin- burgh University). Primers used in the PCR are given in Sup- plementary Table 1. All plasmids and vectors are available from WRAB subject to a materials transfer agreement. Plas- mid DNA was puriﬁed by alkali lysis and precipitation with polyethylene glycol. BACs and PACs were puriﬁed initially by alkali lysis and density gradient centrifugation in mixtures of caesium chloride and ethidium bromide (experiments shown in Figure 4) and subsequently by alkali lysis and PEG precipitation (experiments shown in Figure 8). Cell culture DT40 cells and DT40 somatic cell hybrids were as described (19) and were maintained and electroporated as also described previously (19) except that the medium used was RPMI 1640 including 446 mg/l L-alanyl-L-glutamine with 10% foetal bovine serum, 1% chicken serum, 10 5 M 2-mercapto- ethanol, 10 U/ml penicillin and 10 mg/ml streptomycin. This medium gave cleaner selection after addition of antibi- otics than our earlier DMEM based medium. Targeting of the