Gene inactivation in Lec35.1 (mannosylation-defective) Chinese hamster ovary cells. A cautionary note.
In Lec35.1 CHO mutants, mannose-P-dolichol is synthesized but does not participate in the production of glycosylphosphatidylinositol (GPI) anchor precursors or dolichol-linked oligosaccharides. We tested Lec35.1 cells for stable expression of a cDNA encoding GPI-anchored human folate-binding protein (FBP) with the eukaryotic expression vector pJB20. All normal transfectants, but no Lec35.1 transfectants, expressed FBP activity. However, rather than an inability to produce GPI anchors, lack of FBP expression in Lec35.1 was caused by gradual inactivation of the FBP cDNA. FBP cDNA became fully inactive after 2-3 months of culture, and FBP activity was not restored upon correction of the Lec35 mutation. Southern blot analysis revealed that inactivation was associated with gross rearrangement of FBP cDNA. The cellular FBP gene remained intact. Because the Lec35.1 cell line has the ability to inactivate transfected human FBP cDNA, caution should be exercised when expressing transfected cDNAs in Lec35.1 and similar GPI anchor/glycosylation mutants. Interestingly, these results suggest similarities between the Lec35.1 defect and the human disease paroxysmal nocturnal hemoglobinuria, which may involve gradual inactivation of a gene necessary for mannosylation of GPI anchor precursors.
Slonina
A E
AE
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Scott
T C
TC
Lehrman
M A
MA
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D005786:Gene Expression Regulation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D011956:Receptors, Cell Surface; D014162:Transfection
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D011956:Receptors, Cell Surface; D005492:Folic Acid; D008358:Mannose