Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an invitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS.
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