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

James Ripka, Michael Pierce, Nevis Fregien

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 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 form 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.

Original languageEnglish (US)
Pages (from-to)117-122
Number of pages6
JournalJournal of cellular biochemistry
Volume42
Issue number3
DOIs
StatePublished - Mar 1990

Keywords

  • DNA-mediated transformation
  • Gene transfer
  • GlcNAc-TI
  • Glycosyltransferase
  • Man GlcNAc

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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