Fig. 1: Generation and validation of FMRP knock-out hiPSCs.
From: Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs
A Top: schematic representation of the human FMR1 locus and strategy for gene editing. The red triangle represents the site of cleavage by Cas9. The selection cassette, shown below, encodes for the self-cleavage peptide (T2A) and the puromycin resistance gene (PURO-resistance) and contains a cleavage and polyadenylation signal (pA). Primers used for the PCR analysis on genomic DNA are depicted as arrows. Bottom: PCR analysis on genomic DNA of hiPSCs transfected with the donor construct only (first lane) or co-transfected with the Cas9 and gRNA#1 or gRNA#2 (second and third lanes). B PCR analysis on genomic DNA of the indicated hiPSC clones with primers indicated in panel A. C RT-PCR analysis on RNA isolated from the indicated hiPSC clones with primers spanning exon–exon junctions. The housekeeping gene ATP5O was used as an internal control. D Top: Western blot analysis of FMRP expression in the indicated hiPSC clones. GAPDH was used as loading control. Bottom: immunostaining analysis in parental and FMRP-KO hiPSCs. Red: FMRP; blue: DAPI. Scale bar: 500 μm. E Real-time qRT-PCR analysis of cortical neurons derived from parental and FMRP-KO hiPSCs (n = 3 differentiation batches; Student’s t test; paired; two tails; *p < 0.05, ***p < 0.001). F Western blot analysis of FMRP expression in cortical neurons derived from parental and FMRP-KO hiPSCs. GAPDH was used as loading control.
