The targeted integration of transgenes into a pre-characterized genomic locus enables predictable protein expression to occur, which reduces the need for the screening of transfected clones. We previously developed an accumulative site-specific gene integration system (AGIS), which enabled the repeated integration of multiple transgenes into a pre-determined locus of the cell genome [1, 2]. We achieved the repeated integration of recombinant scFv-Fc gene into the genome of Chinese hamster ovary (CHO) cells, a common animal host cell for the production of recombinant biopharmaceutical proteins. Productivity was shown to correspond to the copy number of the expression cassette . Cell screening after gene transfection was the most time-consuming process in AGIS, but we hypothesized that the use of fluorescent selection markers instead of drug resistant genes would facilitate the cell establishment process because Cre-mediated integration is completed within 48 h post-transfection. Therefore, the present study used marker genes encoding fluorescent proteins to speed up the establishment of producer CHO cells using AGIS.