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We employed a stromal-derived inducing activity (SDIA) model of neurogenesis to investigate the effects of targeted knockdown of Oct-4 and Sox-2 by short interfering RNAs (siRNAs) in mouse embryonic stem (mES) cells. Quantitative real-time PCR showed 40–90% knockdown of specific transcripts with cognate Oct-4 or Sox-2 siRNA transfection compared to FAM-labeled negative control (FAM) siRNA or mock transfection and was confirmed at the protein level by western blot analyses. Upon differentiation using PA6 SDIA co-cultures, neurogenesis is significantly diminished in Oct-4 or Sox-2-targeted mES cells. It was observed that 45 ± 12%, 65 ± 13%, and 90 ± 8% of the colonies were stained with neuron-specific β-tubulin III in Oct-4, Sox-2, and FAM siRNA transfected mES cells, respectively, with similar results observed using neural inducing factors collected from the surface of PA6. Together, our results extend observations for a role of Oct-4 in SDIA and implicate a similar role for Sox-2.