Fig. S6

Ectopic expression of transgenes in zebrafish rods with the aid of Tol2 transposon system. (a) Schematic drawings of Tol2-based expression constructs. Coding sequences of ES1, EGFP and MLS-EGFP were driven by a sequence upstream 1,084 bp of rhodopsin gene ². Each construct was flanked by inverted repeats of Tol2 transposon. Rhup- ES1/EGFP construct was used for the expression of ES1 and a reporter, EGFP, in rods. Rhup-EGFP and Rhup-MLS-EGFP constructs were used as controls. MLS-EGFP contains MLS of COX8 at the N-terminus ³. (b and c) Immunohistochemistries of retinal sections of TG zebrafish harboring Rhup-ES1/EGFP (b, F₀ mosaic fish of the ES1-TG) or Rhup-EGFP (c, F¹ non-mosaic fish of the EGFP-TG) with ES1 antibody (red). EGFP signals are represented in green. Nuclei were stained with DAPI (blue). An isolated rod outer segment and ellipsoid from each retina is shown in each inset. Scale bars, 10 µm in the insets. (d and e) Immunohistochemistries of retinal sections of EGFP-TG (d) or TG harboring Rhup-MLS-EGFP (MLS-EGFP-TG; F₀ generation; mosaic, e) with TOM20 antibody (red). The inset in d and the lower panels in e are magnified views of areas surrounded by dashed lines in d and in the top panel in e, respectively. In panel e, an ellipsoid of MLS-EGFP-TG rod and ellipsoids of wild-type rods are indicated by an arrow and arrow heads, respectively. Scale bars, 10 µm in the main panel of d and the upper panel of e, and 2 µm in the inset of d and the lower panels of e. RE: rod ellipsoid, CE: cone ellipsoid, ONL: outer nuclear layer, INL: inner nuclear layar, GCL: ganglion cell layer.