Figure 3. | Variety of cell types can be epitope-tagged using RNP method.

Representative V5 ICC images are shown for V5 tagging of five TFs that are expressed in both mouse NS cells (A) and ES cells (B). V5 ICC images for the three neural-specific TFs Sox9, Pou3f2, and Pou3f3 in mouse NS cells (Figure C, left panel). Mouse ES cells were electroporated with similar three TF-reagents (Figure C, middle panel). Later, ES bulk populations of non-expressed TFs were differentiated into neural lineage and assayed for expression of TF-V5 fusion proteins by ICC (Figure C, right panel). Differentiation into neural stem progenitors was confirmed by Nestin ICC (in green), only V5-positive rossettes for each of the three genes are shown. (D) V5 ICC images for epitope tagging using human ES cells and GBM patient-derived cell lines.

Figure 3—figure supplement 1. PCR genotyping and sequence confirmation of V5 tagging in mouse ES cells.

(A) PCR strategy is shown, a V5 tag-specific reverse primer and a TF exon-specific forward primer were used to amplify HDR-edited alleles using bulk population of ES cells. (B) Agarose gel analysis of PCR products, gene names are indicated at the top of each lane, ‘- ‘refers to control unedited cells, ‘+’ refers to edited cells, 1kb + DNA ladder is marked with ‘L’. (C) Sequencing trace alignments for some of the edited genes from (b) are shown, Ctcf and Ezh2 genes are expressed in ES cells, Sox9 is expressed after differentiation of ES cells into neuronal lineage.