As stated earlier, Ascl1 was the only gene from the 5F pool that was sufficient to induce neuron-like cells in MEFs. We next attempted to determine the relative contribution of each of the five genes by removing each gene from the pool and assessing the efficiency of iN cell generation. Surprisingly, only the omission of Ascl1 had a dramatic effect on induction efficiency (Supplementary Fig. 6a). Thus, we tested the effects of removing two genes at a time by evaluating all possible three gene combinations. Our results indicated that either Ascl1 or both Brn2 and Myt1l must be present in order to generate iN cells (Fig. 5a). The most efficient conversions were achieved when Ascl1 and Brn2 were combined with either Myt1l (BAM pool) or Zic1 (BAZ pool). The efficiencies in these conditions were two to threefold higher than the 5F pool when the total amount of virus was kept constant (Fig. 5a–d). In this experiment the BAM-iN cells appeared to have a more complex morphology than the BAZ cells (Fig. 5c–d, Supplementary Fig. 7). Therefore, we focused our further analysis on the BAM pool.
