Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A . Schematic diagram showing the genetic structure of the integrated EIAV vectors used in this study . EIAV-CMV-MYO7A (UshStat) is based on a non-replicating non-human recombinant lentiviral vector based on the non-pathogenic wild type equine infectious anaemia virus (EIAV). The wild-type EIAV virus has 6 distinct genetic units, however, the majority of these EIAV sequences have been removed to produce a minimal vector system that contains less than 10% of the original viral genome and does not contain any viral promoters or enhancers and there are no coding regions for accessory proteins in either the EIAV genome or in the packaging system. SIN LTR: Self inactivating long term repeat. Neo: Neomycin open reading frame (ORF). CMV: Cytomegalovirus promoter (constitutive). RK: Rhodopsin kinase promoter (photoreceptor specific). eGFP: enhanced green fluorescent protein ORF. MYO7A: Myosin VIIa ORF. WPRE: Woodchuk hepatitis virus post-transcriptional regulatory element. B . Expression analysis of myosin VIIa in 4 weeks mouse eye and HeLa cells transfected with the EIAV-CMV-Null (Null) or UshStat constructs. β-actin was used as loading control. RPE: retinal pigment epithelium. NR: neuroretina. IP/Null: immunoprecipitates of HeLa cells transfected with the null vector. IP/UshStat: immunoprecipitates of HeLa cells transfected with the myosin VIIa vector. IB MYO7A: immunoblot with the mouse anti-myosin VIIa. Molecular weight markers are denoted to the left. C–D : Immunocytochemistry studies of HeLa cells transduced with the null ( C ) or the UshStat vector ( D ) and immunostained for myosin VIIa (red). DAPI was used to counter stain the nucleus. Scale bar: 15 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Recombinant, Plasmid Preparation, Virus, Expressing, Transfection, Construct, Control, Western Blot, Molecular Weight, Immunocytochemistry, Transduction, Staining

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Shaker1 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). Panel I : 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. Panel II : A–C : Representative examples of an EIAV transduced region of the retina (determine by the presence of GFP), presenting dual immunostaining for GFP and α-transducin ( C ). In the presence of wild type myosin VIIa, α-transducin is translocated to the inner segment (IS). D–F : An untransduced region of the same retina as in A–C does not show α-transducin translocation to the IS upon illumination. G–I : 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 ( H ) however there was no translocation of α-transducin to the IS ( G and I ). 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 D and G 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.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Injection, Plasmid Preparation, Translocation Assay, Expressing, Immunostaining, Transduction, Control

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A–B : Show myosin VIIa immunostaining of shaker1 retinas transduced with the EIAV-CMV-Null vector ( A ) or UshStat ( B ). Myosin VIIa expression can be detected in the RPE, OS and IS regions. C–D : monkey retinas transduced with UshStat ( D ) showed high levels of human myosin VIIa in the RPE and moderate expression in outer and inner segments of the photoreceptor cells. Asterisk in C denotes weak immunostaining of the endogenous myosin VIIa in the RPE layer. Note that the anti-myosin VIIa antibody was titrated so as to only detect the exogenous overexpressed virus-derived wild type myosin VIIa ( B, D ) and not the endogenous protein ( A, C ). Scale bars: 25 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Immunostaining, Transduction, Plasmid Preparation, Expressing, Virus, Derivative Assay

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Retinas were co-transduced with EIAV-CMV-Null vector and EIAV-RK-GFP ( A–C ) or UshStat and EIAV-CMV-GFP ( D–F, J ). After 4 weeks, mice were exposed to 6 days continuous light at 2000 lux illumination. Retinas were harvested and dual immunostained for myosin VIIa (red) and GFP (green) ( A–F, J ). Immunostaining conditions were chosen as to only detect the exogenous overexpressed myosin VIIa. G–H : Eosin and hematoxylin histochemical staining of shaker1 mouse retinas transduced with either the EIAV-CMV-Null vector ( G ) or UshStat ( H ). I : Eosin and hematoxylin staining of wild type (untransduced) retina for comparison of relative light dependent degeneration of the ONL that is typically observed (double headed arrow). J : Low magnification image of an UshStat+GFP co-transduced retina, showing the area used for the studies (bracket) relative to the area of injection (syringe). This area corresponds to the juxtaposed ∼0.2 mm from the site of injection. K : Schematic representation of the area of injection represented by red boxes, where right box area corresponds to the injection in the right eye and left box area injection in the left eye. The inferior retina was always used for the injections and ONL counting. Scale bars A–F : 25 μm, G–I : 50 μm. J : 90 μm. Labels are as in . Asterisks in C , F and J denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Transduction, Plasmid Preparation, Immunostaining, Staining, Comparison, Injection

Journal: bioRxiv

Article Title: In Silico Transcriptome-based Screens Identify Epidermal Growth Factor Receptor Inhibitors as Therapeutics for Noise-induced Hearing Loss

doi: 10.1101/2023.06.07.544128

Figure Lengend Snippet: Afatinib and zorifertinib protect against noise-induced cochlear synaptopathy in mice. ( A ) Representative maximum intensity projections of inner hair cells (IHCs) in the 16-22 kHz region of the cochlea following drug treatment and with (left) or without noise (right). Hair cells were labeled using myosin-VIIa, presynaptic puncta were labeled using CtBP2, and nuclei were counterstained with Hoechst. ( B ) Zorifertinib and Afatinib protect against noise induced cochlear synaptopathy, resulting in less CtBP2 puncta loss with drug + noise than control + noise. The number of CtBP2 puncta per inner hair cell (IHC) is expressed as mean +/- SD; n=2- 5 animals per group. Each dot (n) represents one animal and the average CtBP2 puncta across ten IHCs from two cochleae. *P<0.05. **P<0.01, ***P<0.001; Welch’s ANOVA. Z: Zorifertinib; A: Afatinib; C: Control; N: Noise.

Article Snippet: Antibodies used included: C terminal binding protein-2 (mouse anti-CtBP2; BD Transduction Labs, used at 1:200), myosin-VIIA (rabbit anti-myosin-VIIA; Proteus Biosciences, used at 1:250), anti-otoferlin (HCS-1, DSHB 1:500), anti-GFP (NB100-1614, Novus Biologicals 1:500), total AKT and ERK (Cell Signaling 9272S and 4695S, respectively), phospho forms (AKT-S473 and ERK1/2-T202/Y204, Cell Signaling) and β-actin (Sigma A3854).

Techniques: Labeling, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regeneration of Cochlear Hair Cells and Hearing Recovery through Hes1 Modulation with siRNA Nanoparticles in Adult Guinea Pigs.

doi: 10.1016/j.ymthe.2018.03.004

Figure Lengend Snippet: Figure 3. Supernumerary IHCs Were Uniquely Observed in siHes1 NP-Treated Cochleae (A–F)HCs were immunolabeled withanti-myosin VIIa(green in A, B, E, and F), whereas stereocilia were labeled with fluorophore-conjugated phalloidin (yellow in C–F). Nuclei were stained with DAPI (blue in E and F). Supernumerary IHCs (arrows in B and F) were observed only in noise- deafened OCs treated with siHes1 NPs. Some ectopic IHCs possessed phalloidin-labeled stereocilia (arrowheads in D and F), whereas some presented with no stereocilia (arrows in F). No ectopic HCs were observed in cochleae treated with scRNA NPs (A and E). (G and H) Scanning electron microscope image of an ectopic IHC in an siHes1- NP-treated OC is shown. Ectopic IHCs (arrow in G) with stereociliary structures were also observed by scanning electron microscopy in OCs at three weeks after siHes1 NP treatment (image collected from the 2nd turn of the OC). At this time point, the majority of HCs possessed stereocilia with normal morphology (G). I, P, and O in (G) indicate IHCs, pillar cells, and OHCs, respectively. The scale bars repre- sent 50 mm in (F) for (A)–(F), 10 mm in (G), and 1 mm in (H).

Article Snippet: After blocking, the tissues were incubated with rabbit anti-myosin VIIa, goat anti-prestin (1:20; cat no.: OAEB00391; Aviva Systems Biology, San Diego, CA), and mouse anti-vGlut3 (1:25; cat no.: 135211; Synaptic Systems, Goettingen, Germany) at 4 C overnight.

Techniques: Immunolabeling, Labeling, Staining, Microscopy, Electron Microscopy

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regeneration of Cochlear Hair Cells and Hearing Recovery through Hes1 Modulation with siRNA Nanoparticles in Adult Guinea Pigs.

doi: 10.1016/j.ymthe.2018.03.004

Figure Lengend Snippet: Figure 4. Immature OHCs Were Observed in OCs Treated with siHes1 NPs Immature OHCs were observed in OCs treated with siHes1 NPs by differential prestin immunolabeling at nine (A–H’) and three (I and I’) weeks after treatment. HCs were immunolabeled by anti-myosin VIIa (green in A, B, B’, and G–I’), whereas mature OHCs were immunola- beled with anti-prestin (pink in C–D’ and G–I’). Nuclei were labeled with DAPI (blue in E–I’). Arrowheads in B, D, and H indicate a myosin-VIIa-positive/prestin-negative immature OHC in the 2nd turn of a noise-injured OC treated with siHes1 NPs at nine weeks post- treatment. This immature OHC has an apparently normal OHC morphology (H’). Arrowheads in (I) indicate two myosin-VIIa-positive/prestin-negative OHCs adjacent to a myosin-VIIa-positive/prestin-positive OHC in a noise- injured OC at three weeks post-siHes1 NP treatment. These OHCs were anucleate and small in size (I’). All OHCs in noise-deafened ears treated with scRNA NPs possessed dual labeling with myosin Vlla and prestin (G). The scale bars represent 10 mm in (I) and (I’) and apply to (A)–(I) and (B’), (D’), (F’), (H’), and (I’), respectively.

Article Snippet: After blocking, the tissues were incubated with rabbit anti-myosin VIIa, goat anti-prestin (1:20; cat no.: OAEB00391; Aviva Systems Biology, San Diego, CA), and mouse anti-vGlut3 (1:25; cat no.: 135211; Synaptic Systems, Goettingen, Germany) at 4 C overnight.

Techniques: Immunolabeling, Labeling

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regeneration of Cochlear Hair Cells and Hearing Recovery through Hes1 Modulation with siRNA Nanoparticles in Adult Guinea Pigs.

doi: 10.1016/j.ymthe.2018.03.004

Figure Lengend Snippet: Figure 5. Immature IHCs Were Observed in OCs Treated with siHes1 NPs (A–F) HCs were immunolabeled with myosin VIIa antibodies (green in A, B, G, and H), whereas mature IHCs were differentially immunolabeled with vGluT3 (red in C, D, G, and H). Nuclei were labeled with DAPI (blue in E–H). Arrows in (B) and (H) indicate a myosin-VIIa-positive/vGluT3-negative ectopic immature IHC in a noise-injured OC at nine weeks post-siHes1 NP treatment. Arrowheads in (B), (D), and (H) indicate a myosin VIIa/vGluT3-positive ectopic IHC in the same siHes1-NP-treated OC. All IHCs in OCs treated with scRNA NPS were double labeled with myosin VIIa and vGluT3 (G). The scale bar represents 10 mm in (H) for (A)–(H).

Article Snippet: After blocking, the tissues were incubated with rabbit anti-myosin VIIa, goat anti-prestin (1:20; cat no.: OAEB00391; Aviva Systems Biology, San Diego, CA), and mouse anti-vGlut3 (1:25; cat no.: 135211; Synaptic Systems, Goettingen, Germany) at 4 C overnight.

Techniques: Immunolabeling, Labeling

Journal: Cell and tissue research

Article Title: Expression and localization of somatostatin receptor types 3, 4 and 5 in the wild-type, SSTR1 and SSTR1/SSTR2 knockout mouse cochlea.

doi: 10.1007/s00441-014-1977-7

Figure Lengend Snippet: Fig. 4 Immunofluorescence microscopy detection of SSTR3, -4 and -5 in cultivated NEsc from the OC. Cell nuclei are depicted in blue. The cell membranes of cultivated cells are positive for the HC marker myosin VIIa. a Perinuclear localization of SSTR3 (red), b SSTR4 (red), and c SSTR5 (red) were detected. Scale bar(a–c) 100 μm

Article Snippet: The sections were incubated with primary rabbit polyclonal antiSSTR3, anti-SSTR4, anti-SSTR5 (1:400; AbCam, UK), mouse monoclonal anti-Hes5 antibody (1:300; AbCam) and mouse monoclonal antibody against myosin VIIa (1:500; Abcam).

Techniques: Immunofluorescence, Microscopy, Marker

Journal: Developmental biology

Article Title: Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

doi: 10.1016/j.ydbio.2007.02.028

Figure Lengend Snippet: Fig. 3. Examples of P4.5 Math1-null chimeric and control mice. Panels A–D show HCs in P4.5 mice where β-Gal histochemistry was used to mark the genotypically Math1-null or Math1+/β-Gal cells and myosin VIIa immunocy- tochemistry was used to identify HCs. Double-labeled mutant HCs are shown in the cochlea (arrows in panel A), and in the crista (arrows in panel B) of Math1+/β-Gal chimeras. Similarly double-labeled HCs are also found in the cochlea (C) and crista (D) of Math1-null chimeric mice. Panels E–H show examples of DNA in situ hybridization where labeled cells possess an obvious dark blue/purple dot within the nucleus while the nuclei appear green due to counterstaining with methyl green. The GT probe marks the wild-type HCs (arrowheads in panels E–H), while the mutant HCs remain unlabeled (arrows in panels E–H). All HCs are labeled with DNA in situ hybridization in GT control mice (E), while no HCs are labeled in wild-type mice (F). Examples of genotypically mutant cells are shown in the macula (G) and crista (H) of Math1- null chimeric mice at P4.5. Scale bar=50 μm (in panel D for panels A–D, in panel H for panels E–H).

Article Snippet: The sections were incubated with goat anti-myosin VIIa IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:20 and rabbit anti-β-Gal IgG (Cortex Biochem, San Leandro, CA) at a dilution of 1:25.

Techniques: Control, Labeling, Mutagenesis, DNA In Situ Hybridization

Journal: Developmental biology

Article Title: Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

doi: 10.1016/j.ydbio.2007.02.028

Figure Lengend Snippet: Fig. 4. Examples of Myosin VIIa and β-Gal immunolabeling in HCs in Math1-null chimeric and control mice at P2.5. Images in column 3 (anti-myosin VIIa/anti-β- Gal) are the merged images from column 1 (anti-myosin VIIa) and column 2 (anti-β-Gal). Myosin VIIa is used to label HCs while β-Gal is used to detect cells that are missing at least one copy of the Math1 gene. HCs are labeled only with the anti-myosin VIIa antibody in the cochlea (C) and crista (F) of wild-type mice. Double labeled HCs are shown in the cochlea (arrow in panel I) and crista (arrow in panel L) of Math1+/β-Gal chimeric mice to demonstrate the pattern of intermingling observed in chimeric mice. In Math1-null chimeras, double-labeled mutant cells (arrows in panels O and R) are found intermixed with wild-type cells which are labeled only with anti-myosin VIIa. Double-labeled mutant HCs are shown in the cochlea (arrows in panel O) and crista (arrow in panel R) in the mutant chimeras. The tangential plane of section in panels M–O results in two layers of HCs being visible and the lack of a full view of the HC cytoplasm. Green fluorescence has been found on top of most wild-type and mutant HCs (arrowheads in panels B, E, H, K, N and Q), suggesting an autofluorescence from sterocillia and cuticular plates. Scale bar=50 μm (in panel O for panels A–C, G–I and panels M–O). Scale bar=15 μm (in panel R for panels D–F, J–L and panels P–R).

Article Snippet: The sections were incubated with goat anti-myosin VIIa IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:20 and rabbit anti-β-Gal IgG (Cortex Biochem, San Leandro, CA) at a dilution of 1:25.

Techniques: Immunolabeling, Control, Labeling, Mutagenesis, Fluorescence