FIG. 4.

Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.