FIGURE 3.

Expression of miR-21 and associated changes in TALEN-mediated knockout lines. (A) Complete loss of miR-21expression in three independent lines. The constitutive expression of miR-21 in parental HEK293 cells was high with a CT value of 18∼21. Quantitative PCR confirmed a >1000-fold decrease in miR-21 levels in all three lines tested compared to those of the parental control. (B) No significant change in the mRNA expression level of TMEM49, the closest neighbor of MIR21 on chromosome 17, as assessed by real-time RT-PCR. (C) Expression of miR-21 is associated with corresponding changes in the expression of its targeted gene PDCD4; GAPDH served as loading control. Western blot was conducted with equal amount of proteins extracted from parental HEK293, miR-21 double knockout line, and double knockout line re-expressing miR-21. (D,E) Confirmation of miR-21 re-expression in the miR-21-knockout line. (D) The upper panel shows cells positive for RFP due to presence of the HR donor cassette (bi-allelic). Re-expression of miR-21 was confirmed by co-expression of a GFP marker (lower panel) and real-time RT-PCR (E). (F) Reduced proliferations in all three miR-21 knockout lines tested. Growth curves were plotted for the parental HEK293 cells as well as the three independent knockout lines.