The Cre-loxP system is frequently used for sitespecific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms.

We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins.

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A pharmacopoeia's core mission is to protect public health by creating and making available public standards to help ensure the quality of drugs. In recent years, pharmacopoeias around the world have harmonized their standards in the present context of globalized drug supply chains and markets. For example, the Pharmacopoeial Discussion Group has worked to harmonize excipient monographs and general chapters. In addition, the International Meeting of World Pharmacopoeias has been held by the WHO to discuss information exchange and international collaboration, among other topics. To contribute further to the protection of public health in the globalized drug market, we conducted a comparative study of the pharmacopoeias in Japan, Europe, and the United States. We aimed to examine current differences among the Japanese Pharmacopoeia, the European Pharmacopoeia, and the United States Pharmacopeia-National Formulary and to identify areas that require further collaboration among the three pharmacopoeias. In this study, we analyzed monographs and general chapters listed in the three pharmacopoeias. We identified the features of the monographs and general chapters listed in each pharmacopoeia, as well as differences across the pharmacopoeias. Moreover, on the basis of our findings, we suggest standards that require further collaboration among the pharmacopoeias in certain preferred areas. The comparison data produced by this study are expected to be used to develop strategies for future revisions of pharmacopoeias around the world.