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Antisense and sense cDNA expression cloning using autonomously replicating vectors and toxic lectin selection
L Cummings1, C E Warren, M Granovsky
1Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.
Biochemical and Biophysical Research Communications
|September 15, 1993
Summary
Researchers used toxic lectin ricin to screen for genes that confer resistance in Chinese hamster ovary (CHO) cells. They identified antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) cDNA, demonstrating a novel method for gene discovery.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Chinese hamster ovary (CHO) glycosylation mutant Lec2 cells (CHOP2) are hypersensitive to ricin due to sialic acid deficiency.
- Ricin is a galactose-binding lectin that targets specific glycoconjugates.
Purpose of the Study:
- To identify cDNAs conferring resistance to ricin toxicity in CHOP2 cells.
- To explore the utility of toxic lectin selection for expression cloning.
Main Methods:
- Transfection of CHOP2 cells with a lymphoid cDNA library in pCDM8, followed by selection in ricin-containing medium.
- Expression cloning of cDNAs conferring resistance to ricin.
- Enrichment of sense GlcNAc-TI cDNA in CHOP-1 cells using ConA lectin selection.
Main Results:
- Antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) cDNA was the predominant sequence recovered, conferring a Lec2/Lec1 phenotype.
- Sense GlcNAc-TI cDNA was also enriched in a separate selection experiment.
- Toxic agent-based cell selection proved effective for cloning both sense and antisense cDNA sequences.
Conclusions:
- Antisense GlcNAc-TI expression can alter cellular glycosylation to confer ricin resistance.
- Lectin-mediated cell selection is a powerful tool for expression cloning of functional cDNAs.
- This method facilitates the discovery of genes involved in glycosylation pathways and cellular resistance mechanisms.