Pigv and Dpm1 are simultaneously mutated in clone B502. (A) Functional complementation assay of clone B502 ESCs. Co-transfection of both Dpm1- and Pigv-encoding plasmids was necessary for expression of the GPI-anchored GFP reporter at the cell surface. Original magnification, ×200. Scale bars, 100 μm. (B) Sequence chromatograms of wild-type (upper) and mutant (lower) ESCs at Pigv (left) and Dpm1 (right) loci. Point mutations at the consensus 5′ splice site sequence (GT) of Pigv intron 2 (c.78 + 1G > A) and at the nucleotide next to the consensus 5′ splice site sequence (GT) of Dpm1 intron 7 (c.563 + 3A > T) are shown. (C) Reverse transcription polymerase chain reaction (RT-PCR) of Pigv illustrating that intron 2 is correctly spliced in wild-type but not in mutant ESCs. The arrow indicates a transcript containing an enzymatically important region ; the arrowhead putatively indicates a short isoform. The left lane of the electrophoretic gels represents molecular size markers. Note that cDNA integrity of mutant ESCs was confirmed by amplification of Actb (lower gel). (D) RT-PCR of Dpm1 illustrating that intron 7 is correctly spliced in wild-type (white arrow) but not in mutant ESCs. The left lane represents size markers.