Expression of wild type myosin VIIa in <i>shaker1</i> retinas restores normal thresholds for light-induced α-transducin translocation.

<p><i>Shaker1</i> mouse retinas were co-transduced by a subretinal injection of UshStat and EIAV-CMV-GFP or EIAV-CMV-Null and EIAV-RK-GFP (the GFP vector was used to identify the transduced region of the retina). After 4 weeks the animals were dark-adapted overnight, then light-adapted for 10 minutes under 200 lux illumination. Retinas were double immunostained with antibodies against GFP (green) and α-transducin (red). <b>Panel I</b>: Low magnification image of a retina transduce with UshStat and GFP, showing that the gradient in α-transducin translocation (left to right) parallels GFP expression (indicative of wild type myosin VIIa). Scale bar: 40 μm. <b>Panel II</b>: <b>A–C</b>: Representative examples of an EIAV transduced region of the retina (determine by the presence of GFP), presenting dual immunostaining for GFP and α-transducin (<b>C</b>). In the presence of wild type myosin VIIa, α-transducin is translocated to the inner segment (IS). <b>D–F</b>: An untransduced region of the same retina as in <b>A–C</b> does not show α-transducin translocation to the IS upon illumination. <b>G–I</b>: Retinas co-transduced with EIAV-Null-vector and EIAV-RK-GFP (photoreceptor cell-specific promoter) as a control for the effects of subretinal lentiviral transduction on α-transducin translocation. The region of the retina shown was transduced, evidenced by GFP expression in the photoreceptors (<b>H</b>) however there was no translocation of α-transducin to the IS (<b>G</b> and <b>I</b>). The qualitative results represented in each of the panels are representative images for at least three replicate experiments. RPE = Retinal Pigment Epithelium; OS = Outer Segments; IS = Inner Segments; ONL = Outer Nuclear Layer; OPL = Outer Plexiform Layer. Scale bar: 25 μm. Arrowheads in <b>D</b> and <b>G</b> indicate translocation of α-transducin in individual photoreceptors. Asterisks in I denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.