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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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1316120 | PMC |
http://dx.doi.org/10.1093/nar/gni192 | DOI Listing |
Appl Environ Microbiol
December 2024
MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
Over the past three decades, the integrase (Int) from phage C31 has become a valuable genome engineering tool across various species. C31 Int was thought to mediate unidirectional site-specific integration ( × to and ) in the absence of the phage-encoded recombination directionality factor (RDF). However, we have shown in this study that Int can also catalyze reverse excision ( × to and ) at low frequencies in and , causing genetic instability in engineered strains.
View Article and Find Full Text PDFZoolog Sci
December 2024
Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan,
Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm , TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods.
View Article and Find Full Text PDFSci Rep
November 2024
School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, James Parsons Building, Byrom Street, L3 3AF, Liverpool, UK.
Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL.
View Article and Find Full Text PDFG3 (Bethesda)
October 2024
Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS UMR5239, Université Claude Bernard Lyon 1, 9 rue du Vercors, 69007 Lyon, France.
The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G.
View Article and Find Full Text PDFACS Synth Biol
September 2024
School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
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