Published online 15 December 2005
Methods Online |
Iterative in vivo assembly of large and complex transgenes by combining the activities of 
C31 integrase and Cre recombinase
Institute of Genetics, Queen's Medical Centre, University of Nottingham Nottingham NG7 2UH, UK
*To whom correspondence should be addressed. Tel: +44 115 849 3244; Fax: +44 115 970 9906; Email: William.brown{at}nottingham.ac.uk
Received September 21, 2005. Revised October 17, 2005. Accepted November 25, 2005.
We have used the 
C31 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 
150 kb we have established a method that we call; ‘Iterative Site Specific Integration’ (ISSI). ISSI combines the activities of C31 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.
Present address: Margaret C. M. Smith, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
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