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. 2015 Apr 15;24(14):3956–3970. doi: 10.1093/hmg/ddv134

Figure 1.

Figure 1.

Construction and characterization of mouse and human RPGR-ORF15 AAV vectors. (A) Schematic representation of the vectors. (B) Examination of the vector integrity. PCR amplification of the region spanning the repetitive glutamic acid-glycine coding sequence in the mouse or human RPGR-ORF15 produced the expected 1.3-kb or 1.6-kb fragment in the vector plasmids and all vector preparations. Arrows indicate fragments with minor deletions in two vector preparations. (C) Immunoblot analysis using C100 anti-RPGR antibody that recognizes the C-terminal of mouse RPGR-ORF15 protein. The retinal lysate from an Rpgr-KO mouse injected subretinally with 1 × 109 vg AAV8-mRpgr vector revealed a ∼200-kDa protein band corresponding to the full-length RPGR-ORF15 protein, which is identical to that detectable in a WT C57/Bl6 mouse retina. (D) Immunoblot analysis using an antibody that recognizes the region upstream of the ORF15 exon of human RPGR. The retinal lysate from an Rpgr-KO mouse injected subretinally with 1 × 109 vg AAV8-hRPGR vector revealed a ∼200-kDa protein band corresponding to the full-length RPGR-ORF15 protein (closed arrowheads), identical to that from a commercially sourced human retinal lysate. A set of proteins with lower molecular weights indicating the truncated or alternatively spliced forms of the protein were also detected (open arrowhead). No signal was detected in the lane that included one-tenth amount of retinal lysate from the vector-injected eye, revealing the sensitivity limits of the assay. (E and F) Immunostaining of retina sections from Rpgr-KO mice that received subretinal injection of AAV8-mRpgr (E) or AAV8-hRPGR (F) at 16 weeks post-administration using an antibody against mouse or human RPGR. The magnified images of the marked areas are shown in (E). WT C57/Bl6 and vehicle-injected Rpgr-KO retina sections were used as positive and negative controls, respectively. Vector-expressed RPGR protein mainly localizes to the connecting cilia region of the retina as does the WT protein. RPGR staining is shown in red, and nuclei are stained blue by DAPI. RPE, retinal pigment epithelium; CC, connecting cilia; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 50 µm.