Co-transformation of Lec 1 CHO cells with N-acetylglucosaminyltransferase 1 activity and a selectable marker

In animal cells, the enzyme alpha(1,3)-mannoside-beta(1,2)-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC.2.4.1.101) catalyzes the addition of N-acetylglucosamine to the ASN-linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lec1 is deficient in this enzyme activity and, therefore, accumulates mannose-terminating cell surface ASN-linked oligosaccharides. Consequently, Lec1 cells are sensitive to the cytotoxic effects of the mannose-binding lectin Concanavalin A (Con A). Lec1 cells were co-transformed with human DNA from A431 cells and eukaryotic expression plasmids containing the bacterial neo gene by calcium phosphate/DNA-mediated transformation. Co-transformants were selected for resistance to Con A and G-418. DNA from a primary co-transformant was purified and used to transform Lec1 cells, resulting in secondary co-transformants. Both primary and secondary co-transformants exhibited in vitro GlcNAc-TI-specific enzyme activity. DNA gel blot analysis indicated that secondary co-transformants contained both human and neo sequences.