Figure 3.

Identification of rDNA site-specific gene targeted mESCs. (a) The black arrow indicates a typical mESCs clone after nucleofection with sg1/6-Cas9n and pMrnF9 under selection by G418 for seven days. Scale bar: 200 µm. (b) Twenty-four candidate targeted clones were picked and expanded, and followed by genomic DNA extraction. When amplified with primers F1 and R1, the site-specific integrated clones would obtain a 1679 bp band. M, DL2000 DNA Ladder. (c,d) The PCR products of F1/R1 were sequenced and the results revealed the site-specific integration. (e) Southern Blotting for six candidate targeted clones. Positive clones showed an expected band of 6208 bp. M, DNA Molecular Weight Marker II, DIG labeled. (f) mRNA of four targeted clones were detected by RT-PCR. (g) ELISA for hFIX antigen from the supernatant of targeted mESCs showed the higher level compared with untargeted mESCs (**, p < 0.01). (h) The Neo expression cassette which was flanked by a pair of synclastic FRT elements was removed by secondary nucleofection with the plasmid pCAG-Flpe. When amplified with the primer pair F2 and R2, the excised clones showed a band of 463 bp while 2178 bp indicated non-excision. M, DL2000 DNA ladder. (i) The PCR products of F2/R2 were sequenced and the results showed the excision of the Neo expression cassette. B, Blank, which represented the template was ddH₂O; N, Negative control, which was untransfected.