TARGETED DISRUPTION OF alpha-1, 6-FUCOSYLTRANSFERASE (FUT8) GENE BY HOMOLOGOUS RECOMBINATION IN CHINESE HAMSTER OVARY (CHO) CELLS

摘要

Targeted disruption of both alleles of the alpha-1, 6-fucosyltransferase (FUT8) gene was successful in CHO cells. The double knockout of FUT8 was confirmed by Southern and Northern analyses. The frequency of first-round knockout was estimated as 0.53%of G418-resistant clones, more than ten times that of the second round (0.03%). The functional FUT8 knockout in this cell line was confirmed by its resistance to both LCA and PSA (fucose-recognizing lectins). The HPLC analysis of oligosaccharides in a recombinant monoclonal antibody expressed in this cell line showed a lack of fucose in Asn-linked GlcNAc. The sequential gene-targeting system we used here should be applicable to the modification of properties of recombinant proteins produced by CHO cells.