Using XTN™ TALENs, we were able to target Fut8, the alpha1,6-fucosyltransferase gene, thereby inactivating the fucosylation system and generating a genetically modified CHO cell line for producing afucosylated human therapeutics. Two rounds of transient transfection with expression plasmids encoding XTNs targeting exon 10 of Fut8, combined with a proprietary selection system for nonfucosylated cells, resulted in the generation of a pool of cells in which 80% of the cells lacked fucosylated cell-surface proteins, demonstrated by FACS analysis, and 78% of the Fut8 alleles had been inactivated.
From these pools, clonal cell lines were isolated and further characterized. In Figure 1, using a cell surface detection method, the presence of fucose was assayed using flow cytometry. As expected, the parental CHO isolate demonstrated a clear unimodal population of cells containing fucose (Figure 1A). Likewise, the FUT8 knocked out pools and isolated clones exhibited the expected bi-modal and unimodal populations (Figures 1B and 1C) with the clone showing no detectable fucose on the cell surface.