FIGURE 2.

(A) Direct expression levels of edit specific Aβ are similar for both edited genotypes and dynamic during early stages of differentiation. Stem cell cultures have intermediate expression, embryoid bodies have significantly less expression and differentiated neurons have the highest, reaching maximal expression by ∼10–20 days after EB dissociation and plating in neural differentiation medium. The relative ratios of Aβ expression were ∼1:0.05:5 for the three developmental stages. There were no significant differences in expression level comparing edit specific Aβ40 and Aβ42 at any stage (ANOVA, Dunnett’s correction). No significant secretory Aβ expression was detected in unedited H9 samples. Data were from 6 independent stem cell cultures, 4 EB stage cultures, and 22 individual 10–20-day-old differentiated neuronal cultures. (B) Editing does not affect APP expression from unedited alleles. We used primer pairs spanning three different APP exons. The pattern of expression was similar for all three genotypes and average relative expression for the primer pairs was 1:0.71:0.5 for H9:Aβ40:Aβ42 and is consistent with expected inactivation of one APP due to editing. Expression of edit specific Aβ was ∼30-fold less than APP expression and is replotted from panel (A) for comparison. Data were from 4 independent differentiations of H9 cells and 8–20 differentiations for edited genotypes taken from 10- to 34-day-old cultures. In panel (A) expression was measured by qRT-PCR using a forward primer specific to the secretory signal sequence (not present in the human genome) and reverse primer to a sequence common to Aβ40 and Aβ42. In panel (B) the forward and reverse primers spanned indicated exons in the APP sequence. Bars are mean normalized expression (MNE) relative to GAPDH (±STD).