Figure 3
From: A robust TALENs system for highly efficient mammalian genome editing
Effective miR-210 targeting by our improved TALENs system.
(A) The principle of designing PCR primers that facilitate cell clone screening for potential genomic editing. The 3′ end of the forward or reverse primer should cross the spacer region. (B) PCR screening of 10 SKOV3 cell clones to identify ones with edited miR-210 sequence. Clones 1, 2, 4, 7 and 9, marked by asterisks, produced fewer PCR products than the other five clones. After sequencing, Clones 1, 4, 7 and 9 have successful miR-210 targeting with homozygous editing of miR-210 in Clones 1 and 4. (C) Chromatogram indicates successful homozygous editing of miR-210 sequence in Clone 1 by TALENs as designed in Fig. 2A. Top panel, wild type miR-210 sequence. The highlighted region indicates 22 nucleotides of mature miR-210; lower three panels, truncated miR-210 sequences detected in Clone 1, indicating SKOV3 is aneuploid of chromosome 11. (D) RT-qPCR indicates that miR-210 expression was completely abolished in Clone #1, but parental SKOV3 cells have robust miR-210 expression under 0.5% oxygen (H) and normoxia (N). Arrows in the top panel (amplification plot) indicate the amplification signals from cells with homozygous deletion of miR-210. Arrows in the bottom panel (dissociation curve) indicate that these amplification signals are actually background noises.
