Iterative in vivo assembly of large and complex transgenes by combining the activities of  C31 integrase and Cre recombinase

We have used the [varphi]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...

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Veröffentlicht in:Nucleic acids research 2005-12, Vol.33 (22), p.e189-e189
Hauptverfasser: Dafhnis-Calas, Felix, Xu, Zhengyao, Haines, Steve, Malla, Sunir K, Smith, Margaret C M, Brown, William R A
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container_end_page e189
container_issue 22
container_start_page e189
container_title Nucleic acids research
container_volume 33
creator Dafhnis-Calas, Felix
Xu, Zhengyao
Haines, Steve
Malla, Sunir K
Smith, Margaret C M
Brown, William R A
description We have used the [varphi]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 [varphi]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.
doi_str_mv 10.1093/nar/gni192
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title Iterative in vivo assembly of large and complex transgenes by combining the activities of  C31 integrase and Cre recombinase
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