Antisense and sense cDNA expression cloning using autonomously replicating vectors and toxic lectin selection.
CHOP2 cells, a subline of the Chinese hamster ovary (CHO) cell glycosylation mutant Lec2 which expresses polyoma virus large T antigen, was used as the host cell line to select cDNAs conferring resistance to the toxic effects of ricin. Glycoconjugates in CHOP2 cells are deficient in sialic acid and therefore the cells are hypersensitive to ricin, a galactose-binding lectin. CHOP2 cells acquiring cDNA that either corrected the Lec2 mutation or created a Lec2/Lec1 phenotype were expected to show a selective growth advantage in ricin-containing medium. After a single cycle of transfection with a lymphoid cDNA library in pCDM8 followed by ricin selection, the the predominant cDNA recovered from the cells was antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) which conferred a Lec2/Lec1 phenotype. cDNA encoding GlcNAc-TI in a sense orientation was also enriched by transfecting a cDNA library into CHOP-1 cells, a mutant deficient in this enzyme, and selecting in medium containing ConA lectin. The results show that cell selection with toxic agents can be used to expression-clone both antisense and sense cDNA sequences from bi-directional cDNA libraries in the pCDM8.
Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.
L
L
Cummings
C E
CE
Warren
M
M
Granovsky
J W
JW
Dennis
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D004247:DNA; D004261:DNA Replication; D016373:DNA, Antisense; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis; D008223:Lymphoma; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics; D009844:Oligosaccharides / Q000096:biosynthesis*; D010641:Phenotype; D010957:Plasmids; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D012276:Ricin / Q000633:toxicity*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000952:Antigens, Polyomavirus Transforming; D016373:DNA, Antisense; D006023:Glycoproteins; D009844:Oligosaccharides; D012333:RNA, Messenger; D004247:DNA; D012276:Ricin; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I