Fig. 4

Functional Validation of AAV-based Cre in engineered HEK293 cells. HEK293-TE cells were transfected with the constructs expressing Cre and GFP (a-d). Images were taken at 20× magnifications, 3 days after transduction. Green fluorescence depicts cells expressing Cre-GFP-puro (a). Red fluorescence depicts cells expressing floxed-RFP-puro (b). Overlay of the left and middle images, respectively shows that cells either express RFP or GFP (c), and the phase contract of overlay (d). Alternatively, cells at 20~30% confluency were transduced with the AAV (2.0 MOI) expressing Cre-GFP (e to g) or the control GFP viruses (h to j). Images were taken at 20× magnification, 10 days after transduction Green fluorescence indicates the expression of Cre-GFP (e) or control GFP (h). Diminished red fluorescence indicates the expression of floxed-RFP-puro (f). In contrast, red fluorescence signal remains strong in GFP non-editing control (i). Phase contrast shows the TALEN-Cre sequential edited HEK293 cells (g) vs. control cells (j). White arrows show that GFP and RFP positivity are mutually exclusive. Scale bar 50 μm