Journal: The FASEB Journal

Article Title: Apical CLC‐2 in retinal pigment epithelium is crucial for survival of the outer retina

doi: 10.1096/fj.202100349R

Figure Lengend Snippet: Lentiviral expression of hBest1 promoter CLC‐2‐HA. A‐D, RPE flat mounts from untransduced (control) and lentivirus (LV)‐injected eyes ( LV + ) were labelled with anti‐HA antibody (A‐D). CLC‐2 + /− (A and C) and CLC‐2 −/− RPE flat mounts (B and D) show expression of hBest1 promoter CLC‐2‐HA (green) in transduced eyes (C and D, white arrows indicate injection side), whereas HA‐specific staining is absent in untransduced controls (A and B). Scale bar: 500 µm. E‐F, Retinal cross‐sections from LV‐injected retinas (E and F) show specific localization of hBest1 promoter CLC‐2‐HA (E) on the apical pole of RPE cells, a similar localization pattern as the apical membrane protein MCT1 (F). Transgenic expression of Cetn2 GFP visualizes the photoreceptor transition zone, which is used as reference for orientation in the outer retina. Scale bar: 20 µm

Article Snippet: Lentivirus was generated as previously described., Briefly, 3x10 6 HEK293T cells in DMEM with 10% FBS were transfected with third generation lentivirus (10 μg viral backbone, 3 μg VSV‐G, 5 μg RRE, and 2.5 μg REV) and viral supernatant was concentrated 100‐fold with Lenti‐X Concentrator (Clontech #631231) following the manufacturer’s instructions.

Techniques: Expressing, Control, Injection, Staining, Membrane, Transgenic Assay

Journal: The FASEB Journal

Article Title: Apical CLC‐2 in retinal pigment epithelium is crucial for survival of the outer retina

doi: 10.1096/fj.202100349R

Figure Lengend Snippet: hBest1 promoter CLC‐2‐HA reduces the severity of photoreceptor degeneration and rescues apical localization of MCT1. Retinal cross‐sections from untransduced heterozygous control (A) and untransduced homozygous knockouts (B) show 10‐12 nuclei layers in the ONL are present in control retinas (A, red indicates photoreceptor layer and green the inner nuclear layer) versus the ONL in CLC‐2 −/− mice is degenerated (B, red arrows indicate single photoreceptor nuclei and green the INL). In untransduced RPE flat mounts, MCT1 shows apical staining‐pattern in CLC‐2 + /− mice (D: xz‐view and G), whereas it is largely disrupted in CLC‐2 −/− mice (E: xz‐view and H). Lentivirus was subretinally injected at eye opening (P12) in knockout animals and tissue was harvested at 2M of age. The expression of CLC‐2 under the control of RPE‐specific Bestrophin‐1 promotor (hBest1 promoter ) rescues the retinal degeneration and sustained photoreceptors in mutant mice (C, thickness of the photoreceptor layer, including the OS, is highlighted in red); n = 3. Remarkably, the treatment with LV‐expressing CLC‐2 protein in homozygous knockouts rescues the apical MCT1 staining‐pattern in RPE cells (F: xz‐view and I: lower panel), resembling the distribution pattern of CLC‐2‐HA at the apical membrane surface (I, middle panel); n = 3. Scale bar: 20 µm

Article Snippet: Lentivirus was generated as previously described., Briefly, 3x10 6 HEK293T cells in DMEM with 10% FBS were transfected with third generation lentivirus (10 μg viral backbone, 3 μg VSV‐G, 5 μg RRE, and 2.5 μg REV) and viral supernatant was concentrated 100‐fold with Lenti‐X Concentrator (Clontech #631231) following the manufacturer’s instructions.

Techniques: Control, Staining, Injection, Knock-Out, Expressing, Mutagenesis, Membrane