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. 2016 Jun 9;1:16002. doi: 10.1038/npjregenmed.2016.2

Figure 2.

Figure 2

Knockout of GFP in the axolotl spinal cord NSCs through CAS9–gRNA complex electroporation. (a) DIC (upper panel) and GFP fluorescence (middle panel) images of 6-day regenerates from CAGGS-GFP transgenic axolotls injected with CAS9&GFP–gRNA (left panel) or control (CAS9&Tyr-gRNA, right panel) into the spinal cord lumen and then electroporation. The regenerating spinal cord area, depicted by rectangles, is shown at a higher magnification in the lower panel. Note the clear loss of GFP expression in the regenerating spinal cord tube treated with CAS9&GFP–gRNA as compared with the control. In the CAGGS-GFP transgenic axolotls, GFP is expressed at a much higher level in the mature muscle than in other tissues or in the newly formed immature myofibers. Dotted lines indicate the amputation planes; arrows indicate the spinal cord; dpA, day post amputation. Scale bar, 1 mm. (b) Quantification of the length of 6-day-regenerated spinal cord (solid shapes) and tails (empty shapes) electroporated with CAS9&GFP–gRNA (triangles, n=20) or control CAS9&Tyr-gRNA (Ctr, diamonds, n=12). Error bars, s.d. (c) GFP fluorescence (green) and immunofluorescence for SOX2 (red) combined with DAPI (blue) on 10-μm cross-cryosections show the massive loss of GFP expression in NSCs along the spinal cord from mature ('M') to regenerated ('R') regions of 6-day tail regenerates treated with CAS9&GFP–gRNA compared with the control. The spinal cord areas (squares) are showed at higher magnification as single channel or merged images. Scale bar, 200 μm. (d and e) Quantification of (d) the number of nuclei per cross-section in the tubular spinal cord and (e) the percentage of GFP+ SOX2+ cells over total SOX2+ spinal cord cells of 6-day regenerates at different positions along their length from axolotls treated with CAS9&GFP–gRNA (triangles, n=5) or CAS9&Tyr-gRNA control (diamonds, n=5). Each scale on the x-axis represents ~800 μm of length. M, mature uninjured tail region anterior to the amputation plane; R, regenerate posterior to the amputation plane. Error bars, s.d.; ***P<0.001.