FIGURE 2. Mouse ES cells with heterozygous deletion of Rest or mouse ES cells treated with siRest differentiate into multiple lineages.

a, b, RT–PCR (a) and qRT–PCR (b) analyses of total RNA isolated from ES and YHC cells grown under self-renewing conditions were performed using primers specific for markers for ectoderm, mesoderm, endoderm and trophectoderm. Gapdh was used as an internal control. *, P = 0.001; **, P = 0.003; ***, P = 0.006; ****, P = 0.012. The values are represented as mean  s.d. (n = 3). c, EB cells derived from YHC cells show lineage markers that are either absent or significantly reduced in EB cells derived from wild-type mouse ES cells. RT–PCR analysis of total RNA isolated from wild-type EB cells and YHC EB cells was performed using specific primers for various differentiated lineage genes (shown above each lane). Gapdh was used as a loading control. d, qRT–PCR analysis of total RNA isolated from mouse ES cells treated with either non-targeting siRNA (NT) or siRest was performed using primers specific for different lineage markers. Analysis was performed 5 days after transfection. *, P < 0.0001; **, P = 0.001; ***, P = 0.002; ****, P = 0.004; *****, P = 0.005; ******, P = 0.01. The values are represented as mean  s.d. (n = 3). e, The inner cell mass of blastocysts showed co-expression of REST and self-renewal markers Oct4, Nanog and Sox2. Blastocysts were stained for nuclei (4,6-diamidino-2-phenylindole (DAPI), blue), REST (red) and self-renewal markers (green). Merged and differential interference contrast (DIC) images are also shown. Scale bar, 25 m.