Knockout of the miR-9-2 gene in H9 cells.

<p>(<b>A</b>) Schematic representation of the miR-9-2 gene and a 100-mer ssODN used for miR-9-2 deletion. The box in the miR-9-2 locus indicates the stem-loop structure of the gene. Homology regions between the ssODN and the miR-9-2 gene are indicated. (<b>B</b>) Genomic PCR screening of the miR-9-2 gene deletion. H9 clones transfected with the TALEN and ssODN were isolated. Genomic PCR to amplify the region surrounding the TALEN cutting site followed by BamH1 digestion was carried out. The expected fragment sizes for wild-type and ssODN-mediated HDR are 373 bp and 284 bp, respectively. BamHI cuts the 284 bp fragment into 213 bp and 69 bp fragments. (<b>C</b>) Immunofluorescence staining of stem cell markers in undifferentiated clone 2247 cells. Nuclei were counterstained by DAPI. Scale bar = 100 μm. (<b>D</b>) Immunofluorescence staining of TUJ1 in neuronal cells derived from differentiated H9 and clone 2247 cells. Scale bar = 100 μm. (<b>E</b>) Level of mature miR-9 in H9 and clone 2247. Quantitative RT-PCR was used to measure the level of mature miR-9. Day 0 denotes the undifferentiated cells whereas day 45 denotes neuronal cells. <i>p</i><0.05. (<b>F</b>) Level of precursor miR-9-1 and miR-9-2 in H9 and clone 2247. Pre-miR-9-2 was undetectable in clone 2247.