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. Author manuscript; available in PMC: 2015 Nov 19.
Published in final edited form as: Cell Stem Cell. 2014 Apr 3;14(6):781–795. doi: 10.1016/j.stem.2014.03.004

Figure 2. Genetic Correction of the SOD1A4V Mutant Allele Rescues Motor Neuron Survival and Soma Size Deficits.

Figure 2

(A) Gene targeting strategy used to generate isogenic 39b-SOD1+/+ iPSC line. Nucleases targeting the SOD1 locus created a double strand break upstream of Exon1; homologous recombination of the genomic locus with a targeting plasmid with control sequence of Exon1 coupled to PGK∷Puromycin replaced the SOD1A4V mutant allele; after antibiotic selection, the resistance cassette was removed by Flp-mediated recombination, leaving only an FRT footprint.; ZFN: Zinc Finger Nuclease, FRT: Flippase Recognition Target. (B) Sequencing chromatograms of Exon1 of SOD1 in iPSC line 39b before and after targeting, demonstrate the correction of the A4V mutation. (C) PshAI restriction digestion of amplified SOD1 cDNA before and after gene targeting. The mutation creates a PshAI restriction site that is absent in the corrected line. (D) qPCR of genomic SOD1 shows that there are no extra copies of the gene in the corrected line and (E) qRT-PCR of cDNA shows that the expression levels of SOD1 are the same in the corrected line (n=3, +/-SEM). (F) SOD1 protein levels assessed by western blot (WB) analysis in parental (39b-SOD1+/A4V), targeted hemizygous knockout (39b-SOD1+/-) and corrected (39b-SOD1+/+) iPSC clones. (G) Isogenic 39b-SOD1+/+ MNs exhibit increased cell survival (n=6, m>13000, +/-SEM, P<0.05) and (H) soma size (n=3, m=280, +/-SEM, P<0.01). (I) WB analysis of SOD1 protein in detergent- soluble (RIPA) and detergent-insoluble (UREA) fractions in day 12 neuronal cultures. Insoluble SOD1 is not detected under normal conditions. After proteasome inhibition by MG132 treatment, insoluble SOD1 selectively accumulates only in SOD1+/A4V MNs and not in the corrected line.