Genetically Engineered CHO Cell Lines

HEK293T cells (ATCC, cat# CRL-3216) used in this study were cultured in Dulbecco’s Modified Eagle Medium, DMEM, supplemented with 10% FBS, 1% NEAA and 1% L-Glutamine at 37 °C and 5% CO₂. Genetically engineered CHO cells, B4GalT7 KO, Csgalnat1/2/Chsy1 KO, Extl3 KO, Ndst1/2 KO, Hs2st1 KO, Glce KO, Hs6st1-3 KO, NDST2 KI, HS3ST1 KI Csgalnact1/2/Chsy1 KO, HS3ST5 KI Csgalnact1/2/Chsy1 KO, CHST3 KI, CHST15 KI were maintained in suspension culture using an equal mix of EX-CELL® CD CHO Serum-Free Medium (#14360 C, Sigma-Aldrich) and BalanCD CHO Growth A medium (#91128, Fujifilm), supplemented by 2% GlutaMAX™ (#35050061, Gibco). These CHO cell lines were generated and are maintained in the Copenhagen Centre of Glycomics, the cell plasmids and gRNA constructs designed for each CHO cell are described in refs. ^{33 (link),34 (link)}. Protein expression was induced by transfection with plasmid DNA and polyethyleneimine as the transfection reagent.