Experimental design. (A) Biosynthetic pathway of GPI-anchored proteins. GPI-anchor precursors are synthesized and attached to proteins in the endoplasmic reticulum (ER). The resulting GPI-anchored proteins are expressed at the cell surface. Out of 26 genes involved in this pathway, four (shown in red) were excluded from our screening (see text). Dol-P-Man, dolichol-phosphate-mannose. (B) Schematic screening procedure. (C) Functional complementation assay. Upper, principle of the assay. Lower, representative culture fields of Piglm mutant ESCs unrestored (left) and restored (right) by plasmid transfection (scale bars, 50 μm). For clarity, original images shown in Additional file 1: Figure S1C were trimmed.