(A) Schematic diagram of the gene targeting strategy at the mouse Actb locus. A donor vector containing a promoter-less p2A-puromycin (Puro) and GFP gene driven by the CAG promoter was designed for targeting the mouse Actb locus. The underlined trinucleotide represents the PAM, and the sgRNA targeting site is labeled in red. The length of the left and right homologous arm is 801 bp and 800 bp, respectively. (B) Representative images of Coomassie blue stained puromycin resistant E14 cells which were successfully knocked in. Transfection of donor only or the mixture of sgRNA and Cas9 were set as the negative control. (C) Representative microscopy images of successfully targeted E14 cells with GFP expression. (D) Effect of different small molecules on gene targeting frequency at the Actb locus in mouse ESCs. The knock-in efficiency was measured by counting the cell colonies which were resistant to puromycin. (E) The Sanger sequencing results of the 5’ and 3’ junction regions of successfully knocked-in cells treated with farrerol. HAL stands for the left homologous arm and HAR stands for the right homologous arm. Error bars represent the s.e.m. **p<0.01, ***p<0.001, n.s., not significant, t-test. All experiments were repeated at least three times.
Figure 3—source data 1. Summary of knock-in efficiency in mouse ESCs.