Fig 2. Analysis of PIG-A gene expression in human ES cells lacking GPI-APs.

(A). Conventional RT-PCR of the PIG-A gene expression in undifferentiated ES cells (undiff. ES) and differentiated hES cells after BMP4 induction or embryoid body formation (EB) after 10 days. G-GFP hES cells (samples 1) and AR1-c1 (samples 2) clonal hES cells were analyzed side by side. In addition, we analyzed reconstituted AR1-c1 hES cells after transduction of PIG-A transgene that restored GPI-AP expression (samples 3). (B). Similar analysis of AR2-c1 hES cells (sample 4) and the AR2-c1 cells restored by the PIG-A transgene expression (sample 5). (c) Quantitative RT-PCR analysis of the PIG-A gene in AR1-c1 hES cells before and after differentiation. The relative level of PIG-A mRNA is first normalized by that of beta-actin, and then by the level in teratoma (defined as 100), which was used as a common positive control in quantitative RT-PCR analyses. The mean and SD (n=4) were plotted in a log scale. The PIG-A mRNA in undifferentiated G-GFP cells is found at a low level (1-2% of teratoma), but elevated significantly after differentiation by BMP4 induction or EB formation (A and C-D). Notably, the PIG-A mRNA level in undifferentiated AR1-c1 hES cells (sample 2) and AR2-c1 (sample 4) was much lower (∼ 10 fold) than the control G-GFP cells by both assays in (A-C). The deficiency of PIG-A mRNA in AR1-c1 cells is more obvious after differentiation (A, C). (D). Northern blot of PIG-A mRNA in AR1-c1 cells confirmed RT-PCR data that the PIG-A deficiency is due to the lack of PIG-A mRNA.