Generation of a human haploid neural stem cell line for genome-wide genetic screening

Figure 5 PiggyBac transgenesis in the derived haploid neural stem cells. A: Schematic overview of PiggyBac (PB) insertion. PB transposons were inserted into the genomes of derived haploid neural stem cells (haNSCs) with the help of PBase; B: Bright field (left) and FITC (right) images of haNSCs after transfection. Scale bar, 100 μm; C: Schematic of the splinkerette PCR. 5’ and 3’ PB-void BstYI was used to digest genomic DNA from derived haNSCs, and T4 DNA ligase was used to form the LEFT and RIGHT cycle. After ligation, the insertion sites were verified by two rounds of nested PCR. The primers are shown as black arrows and listed in Supplementary Table 1; D: Agarose gel electrophoresis was used to analyze the 5’ inverted terminal repeat PCR products harvested from transgenic haNSCs; E: Illustration of the sense and antisense; F: Distribution of insertion sites in the genome; G: Gene Ontology enrichment analysis; H: Top 10 most frequently inserted genes in the PB transposon-based mutagenesis.