(A) A complex N-glycan with glycosyltransferases responsible for each reaction. Combinatorial knockout of the glycosyltransferase isoenzyme genes indicated led to the identification of the primary genes (highlighted in bold) controlling N-glycan branching (Mgats), elongation (B3gnts and B4galts), and sialylation (St3gals) in Chinese hamster ovary (CHO) cells. The methods discussed are equally applicable to the glycosyltransferases responsible for other types of glycosylation. (B) A generalized scheme for genetically altering the expression of different classes of glycans on cells. Gene deletions, insertions, or activation of glycosyltransferases can be used to generate isogenic cell lines that display different glycan features on endogenous surface glycoconjugates. Libraries of such engineered isogenic cells can be used to determine binding specificities of lectins, toxins, antibodies, or viruses. For illustration, isogenic cells with selective loss of different types of glycans are shown (loss of elaborated N-glycans [KO MGAT1], O-glycans [KO C1GALT1], and glycosphingolipids [KO B4GALT5/6]).