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. 2019 Jan 30;33(4):5287–5299. doi: 10.1096/fj.201801110RR

Figure 8.

Figure 8

Impaired network firing in SCN2A+/− iN networks. A) Schematic illustration of the strategy for establishing SCN2A+/− hESCs using CRISPR/Cas9 genome editing. The locations of sgRNAs targeting the human SCN2A gene are indicated by blue arrows (top panel) and highlighted in light blue, with the corresponding protospacer-adjacent motif (PAM) in red (bottom panels). Red arrowheads indicate predicted double-stranded break (DSB) sites. B) Sequencing results from 2 monoclonal hESC lines (SCN2A+/−-1 and SCN2A+/−-2) produced with sgRNA-1 and sgRNA-2, respectively. Red arrowheads indicate predicted DSB sites. Mutant sequences are highlighted in red. Uppercase sequence indicates exons, and lowercase sequence indicates introns. C) The mRNA expression levels of SCN2A in the 2 SCN2A+/− lines, normalized to the expression of SCN2A in WT iNs. D) Quantification of the mean spike rate of WT, SCN2A+/−-1, and SCN2A+/−-2 iN networks at different time points during development. Data were collected from 9 MEAs for each line. E) Representative raster plots of network activities of WT and SCN2A+/−-1 iN networks recorded by MEA at d 60 in the absence and presence of PTX (20 μM) treatment. F) Analysis of the fold change in the spike rate for WT and SCN2A+/− iN networks in response to PTX (20 μM) treatment. Data were collected from 9 MEAs for each line. N,s., not significant. **P < 0.01 (unpaired Student’s t test).