Co-transformation of Lec1 CHO cells with N-acetylgucosaminyltransferase 1 activity and a selectable marker

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Abstract
In animal cells, the enzyme α( 1,3)‐mannoside‐β( 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 Lecl is deficient in this enzyme activity and, therefore, accumulates mannose‐terminating cell surface ASN‐linked oligosaccharides. Consequently, Lecl cells are sensitive to the cytotoxic effects of the mannose‐binding lectin Concanavalin A (Con A). Lecl 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 Lecl 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.