GNA probe for cell surface oligomannose glycan expression.

GNA binds glycan structures with terminal mannose and will not bind complex, sialic acid–containing glycans. CHO-S cells were transfected with a plasmid designed to inactivate the MGAT1 gene by CRISPR/Cas9 gene editing (MGAT CHO). The cells were treated with fluorescein-conjugated GNA to screen for the incorporation of high-mannose glycans in the cell membrane. HEK 293 GnTI− cells that also lack the MGAT1 gene served as a positive control (panels A and D), while normal CHO-S cells that possess an intact MGAT1 gene served as a negative control (panels B and E). Cells were visualized under 20× magnification on a Leica DM5500 B widefield microscope using DIC (upper panels A, B, C) or under illumination with 495-nm light (lower panels D, E, and F). Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeat; DIC, differential interference contrast; GNA, Galanthus nivalis lectin; GnTI, N-acetylglucosaminyltransferase I; HEK 293, human embryonic kidney 293; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase.