title: Iterative in vivo assembly of large and complex transgenes by combining the activities of phiC31 integrase and Cre recombinase.
abstract: We have used the phiC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than approximately 150 kb we have established a method that we call; 'Iterative Site Specific Integration' (ISSI). ISSI combines the activities of phiC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.
authors.affiliation: Institute of Genetics, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.
authors.forename: Felix
authors.initials: F
authors.lastname: Dafhnis-Calas
authors.forename: Zhengyao
authors.initials: Z
authors.lastname: Xu
authors.forename: Steve
authors.initials: S
authors.lastname: Haines
authors.forename: Sunir K
authors.initials: SK
authors.lastname: Malla
authors.forename: Margaret C M
authors.initials: MC
authors.lastname: Smith
authors.forename: William R A
authors.initials: WR
authors.lastname: Brown
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000201:enzymology; D002460:Cell Line; D002645:Chickens / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases / Q000378:metabolism*; D011995:Recombination, Genetic; D013302:Streptomyces / Q000821:virology; D020080:Tandem Repeat Sequences; D019076:Transgenes; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases