Journal: EMBO Molecular Medicine

Article Title: Effective delivery of large genes to the retina by dual AAV vectors

doi: 10.1002/emmm.201302948

Figure Lengend Snippet: A–F Western blot analysis of HEK293 cells infected with AAV2/2 vectors encoding for ABCA4 (A, D), MYO7A (B, E) and EGFP (C, F). The Western blot images (A-C) are representative of and the quantifications (D-F) are from n = 4 (A, C, D, F) or n = 3 (B, E) independent experiments. OZ, AAV oversize; OV, dual AAV overlapping; TS, dual AAV trans-splicing; AP, dual AAV hybrid AP; AK, dual AAV hybrid AK; TS-L, dual AAV trans-splicing EGFP with a combined genome size similar to OZ-EGFP; AK-L, dual AAV hybrid AK EGFP with a combined genome size similar to OZ-EGFP; 5′+3′, cells co-infected with 5′-and 3′-half vectors; 5′, control cells infected with the 5′-half vector; 3′, control cells infected with the 3′-half vector; α-EGFP, Western blot with anti-EGFP antibody; α-3xflag, Western blot with anti-3xflag antibody; α-Myo7a, Western blot with anti-Myo7a antibody; α-β-Tubulin, Western blot with anti-β-Tubulin antibody, used as loading control; α-Filamin A, Western blot with anti-Filamin A antibody, used as loading control. * P ANOVA < 0.05; ** P ANOVA < 0.001. A–C The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left. D–F Quantification of ABCA4 (D), MYO7A (E) and EGFP (F) protein bands. The intensity of the ABCA4, MYO7A and EGFP bands was divided by the intensity of the Filamin A (D, E) or Tubulin (F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). Data information: (E) The asterisks represent significant differences with both OZ and AP. (D–F) More details on the TS and TS-L variability as well as on the statistical analysis including specific statistical values can be found in the Western blot and Statistical analysis paragraphs of the Materials and methods section, respectively.

Article Snippet: The antibodies used for immuno-blotting are as follows: anti-EGFP (1:500, sc-8334; Santa Cruz, Dallas, TX, USA); anti-3xflag (1:1000, A8592; Sigma-Aldrich); anti-Myo7a (1:500, polyclonal; Primm Srl, Milan, Italy) generated using a peptide corresponding to aminoacids 941–1070 of the human MYO7A protein; anti-HA antibody (1:2000, PRB-101P-200, HA.11; Covance, Princeton, NJ, USA); anti-β Tubulin (1:10 000, T5201; Sigma Aldrich); anti-Filamin A (1:1000, catalog#4762; Cell Signaling Technology, Danvers, MA, USA); anti-Dysferlin (1:500, Dysferlin, clone Ham1/7B6, MONX10795; Tebu-bio, Le Perray-en-Yveline, France).

Techniques: Western Blot, Infection, Plasmid Preparation, Molecular Weight, Expressing

Journal: EMBO Molecular Medicine

Article Title: Effective delivery of large genes to the retina by dual AAV vectors

doi: 10.1002/emmm.201302948

Figure Lengend Snippet: Representative Western blot analysis of C57BL/6 eyecups 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 μg of proteins were loaded in each lane. The number ( n ) and percentage of MYO7A-positive eyecups out of total eyecups analyzed is depicted. AK, eyes injected with dual AAV hybrid AK vectors; TS, eyes injected with dual AAV TS vectors; neg, eyes injected with either 5′-or 3′-half of the dual AAV TS and hybrid AK vectors; α-HA, Western blot with anti-hemagglutinin (HA) antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control. Quantification of MYO7A levels expressed from dual AAV vectors in sh1 −/− eyecups relative to endogenous Myo7a expressed in littermate sh1 +/– eyecups. sh1 −/− eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of the CBA promoter and analyzed 1.5 months later. sh1 +/– eyes were injected with AAV vectors expressing EGFP. The number ( n ) of eyes analyzed is depicted below each bar. The quantification was performed by Western blot analysis using the anti-Myo7a antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin. The histograms show the expression of MYO7A protein as percentage relative to sh1 +/– Myo7a; the mean value is depicted above the corresponding bars. Values are represented as mean ± standard error of the mean (s.e.m.).

Article Snippet: The antibodies used for immuno-blotting are as follows: anti-EGFP (1:500, sc-8334; Santa Cruz, Dallas, TX, USA); anti-3xflag (1:1000, A8592; Sigma-Aldrich); anti-Myo7a (1:500, polyclonal; Primm Srl, Milan, Italy) generated using a peptide corresponding to aminoacids 941–1070 of the human MYO7A protein; anti-HA antibody (1:2000, PRB-101P-200, HA.11; Covance, Princeton, NJ, USA); anti-β Tubulin (1:10 000, T5201; Sigma Aldrich); anti-Filamin A (1:1000, catalog#4762; Cell Signaling Technology, Danvers, MA, USA); anti-Dysferlin (1:500, Dysferlin, clone Ham1/7B6, MONX10795; Tebu-bio, Le Perray-en-Yveline, France).

Techniques: Western Blot, Injection, Molecular Weight, Expressing

Journal: EMBO Molecular Medicine

Article Title: Effective delivery of large genes to the retina by dual AAV vectors

doi: 10.1002/emmm.201302948

Figure Lengend Snippet: Semi-thin retinal sections representative of both sh1 +/+ and sh1 +/– eyes ( sh1 +/+ ) injected with AAV vectors expressing EGFP and of sh1 −/− eyes injected with dual AAV trans-splicing (TS- MYO7A ), hybrid AK (AK- MYO7A ) or the 5′-or 3′-half vectors (neg), as negative controls. The arrows point at correctly localized melanosomes, the scale bar (10 μm) is depicted in the figure. Quantification of melanosome localization in the RPE villi of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes analyzed is depicted below each bar. The quantification is depicted as the mean number of apical melanosomes/100 μm, the mean value is depicted above the corresponding bar. sh1 −/− neg includes sh1 −/− eyes injected with AAV vectors expressing either 5′-( n = 1) or 3′-( n = 2) half of the dual TS vectors, or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05, ** P ANOVA < 0.001. Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes and connecting cilia analyzed is depicted below each bar. sh1 −/− neg includes sh1 −/− eyes injected with AAV vectors expressing either 5′-half of the dual TS vectors ( n = 3) or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). The quantification is depicted as the mean number of gold particles per length of connecting cilium, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.

Article Snippet: The antibodies used for immuno-blotting are as follows: anti-EGFP (1:500, sc-8334; Santa Cruz, Dallas, TX, USA); anti-3xflag (1:1000, A8592; Sigma-Aldrich); anti-Myo7a (1:500, polyclonal; Primm Srl, Milan, Italy) generated using a peptide corresponding to aminoacids 941–1070 of the human MYO7A protein; anti-HA antibody (1:2000, PRB-101P-200, HA.11; Covance, Princeton, NJ, USA); anti-β Tubulin (1:10 000, T5201; Sigma Aldrich); anti-Filamin A (1:1000, catalog#4762; Cell Signaling Technology, Danvers, MA, USA); anti-Dysferlin (1:500, Dysferlin, clone Ham1/7B6, MONX10795; Tebu-bio, Le Perray-en-Yveline, France).

Techniques: Injection, Expressing

Journal: PLoS Biology

Article Title: Mouse screen reveals multiple new genes underlying mouse and human hearing loss

doi: 10.1371/journal.pbio.3000194

Figure Lengend Snippet: ABR thresholds of Klc2 mutants are close to those of controls at 2 weeks old, and show significant, progressive increases in thresholds from one month onwards, mainly at low frequencies (3–18 kHz), increasing to affect higher frequencies by 6 months old (A-E) (wild types 2 weeks n = 4, 1 month n = 13, 2 months n = 13, 3 months n = 9, 6 months n = 15; Klc2 homozygotes 2 weeks n = 6, 1 month n = 13, 2 months n = 13, 3 months n = 13, 6 months n = 18; Mann–Whitney U test, at 2 weeks old, p = 0.12 overall but p = 0.019 at 3 kHz and p = 0.01 at 6 kHz when testing stimuli separately; p < 0.001 at each later age shown by asterisks). F. DPOAEs at 6 months old show mutant amplitudes (red, n = 3) at the noise floor (grey) across all frequencies tested; wild types (green, n = 3) show normal emission amplitudes. Gi-vi. Scanning electron microscopy revealed extensive loss of OHC hair bundles at P28 in the cochlear regions, corresponding to the worst thresholds in mutants (12 kHz; Gi. heterozygote n = 4, Gii. homozygote n = 11), while there was little sign of hair cell loss at higher-frequency regions (42 kHz; Giii. heterozygote, Giv. homozygote). Remaining hair bundles had a normal appearance (Gv. heterozygote, Gvi. mutant). Scale bars, g-j, 10 μm; k-l, 1 μm. Hi-iv. Confocal imaging at P28 showed that many OHC nuclei were missing in the most affected regions (12 kHz; Hi. heterozygote n = 4, Hii. homozygote n = 5), but most hair cell nuclei were present at less affected regions (30 kHz; Hiii. heterozygote, Hiv. homozygote). Blue, DAPI-labelled nuclei; red, CtBP2-labelled ribbons and IHC nuclei; green, neurofilament-labelled unmyelinated dendrites. Scale bars, 10 μm. Hv. Quantification of OHC nuclei from confocal images demonstrated significant reduction in mutants (red) at best-frequency regions from 6 to 24 kHz and no significant difference with controls (green) at the 30-kHz region. Black line represents ABR threshold elevation in mutants compared with littermate controls. I. EPs in wild types (green) and homozygotes (red) show no significant difference in mutants (homozygotes 113.8 ± 11.5 mV, n = 10; wild-type littermates 116.0 ± 6.5 mV, n = 10; t = 0.518, df = 14, two-tailed p -value = 0.613). Maximum negative potentials during anoxia are significantly reduced in homozygotes (lower part of plot) (homozygotes −10.0 ± 5.39 mV, n = 6; wild-type littermates −33.7 ± 4.8 mV, n = 6; t = −8.050, df = 10, two-tailed p -value = 0.0000111). Ji-vi. Confocal imaging of IHCs at P28 showed no obvious abnormalities of GluR2-labelled postsynaptic densities (green), but less extensive Kcnma1-labelled patches (red) of IHCs in mutants compared with controls at the frequency regions showing the worst thresholds (6 and 12 kHz) ( n = 7 homozygotes, 5 littermate controls). Scale bars, 5 μm. Jvii. Counts of green-labelled GluR2 puncta per IHC show no difference between mutants and wild types (6 kHz, homozygotes n = 7, wild types n = 5, t test, p = 0.7422; 12 kHz, homozygotes n = 7, wild types n = 5, t test, p = 0.0737; 30 kHz, homozygotes n = 5, wild types n = 6, t test, p = 0.7089). Ki. Representation of the allele, with exons in grey, FRT sites in green, loxP sites in red, and lacZ and neo components of the inserted construct labelled. Kii-iii. Expression of Klc2 in the cochlear duct of a heterozygote ( n = 3) using the LacZ reporter system in the allele. Blue-labelled areas show expression in cells surrounding the cochlear duct and spiral ganglion. Kiii shows a higher magnification of the organ of Corti. Scale bar on Kii, 100 μm, on Kiii, 50 μm. Li-ii. Confocal images of the organ of Corti in a wild type (left) and homozygote (right) at P28 labelled with Myo7a antibody (false-coloured green) showing hair cell bodies and DAPI (false-coloured red) showing nuclei. Small images at left and right show the areas marked in white boxes rotated through 90° around the radial cochlear axis to show a mid-modiolar view of the hair cells. Nuclei appear in similar locations in mutants and controls (base of OHCs, middle of IHCs). Scale bars, 5 μm. M. qRT-PCR of Klc2 mRNA from brain at P28 showed complete knockdown of transcript in homozygotes (red, n = 3); heterozygotes (blue, n = 4) showed around half of the wild-type level (green, n = 2). All plots are means ± standard deviation. Plotted data points are given in . ABR, auditory brainstem response; DPOAE, distortion product otoacoustic emission; EP, endocochlear potential; FRT, flippase recombinase target; IHC, inner hair cell; lacZ , gene encoding β-galactosidase; loxP, locus of crossover in P1 bacteriophage; OHC, outer hair cell; P28, postnatal day 28; qRT-PCR, quantitative real-time PCR.

Article Snippet: The primary antibodies used overnight at RT were mouse anti-GluR2 (1:200, MAB397, Emd Millipore), rabbit anti-Kcnma1 (1:100, APC-021, Alomone), rabbit anti-Myo7a (1:200, PTS-25-6790-C050, Axxora), mouse anti-Ctbp2 (1:400, BD Transduction Laboratories 612044), and chicken anti-NF-H (1:800, Abcam ab4680).

Techniques: MANN-WHITNEY, Mutagenesis, Electron Microscopy, Imaging, Two Tailed Test, Construct, Expressing, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: JARO: Journal of the Association for Research in Otolaryngology

Article Title: Quantitative Analysis of Aquaporin Expression Levels during the Development and Maturation of the Inner Ear

doi: 10.1007/s10162-016-0607-3

Figure Lengend Snippet: Detailed distribution of Aqp11 mRNA in the organs of Corti on P3 (a–c) and P21 (d–f). In situ hybridization for Aqp11 (a and d), immunohistochemistry for Myo7a (b and e), and merged images (c and f) are shown. Schematic illustrations indicate cells expressing Aqp11 on P3 (g) and P21 (h). On P3 (a–c) and P21 (d–f), Aqp11 mRNA was detected in the OHCs (magenta arrowheads), pillar cells (magenta open arrows), Deiter’s cells (magenta arrows), and Hensen’s cells (magenta open arrowheads), as schematically illustrated (g and h). Abbreviations: i, IHC; o, OHC; p, pillar cell; d, Deiter’s cell; h, Hensen’s cells. Bars indicate 50 μm.

Article Snippet: After blocking with 10 % goat-serum in PBS:0.2%Triton-X, rabbit polyclonal anti-Myo7a antibody (1:1000, GeneTex, USA) was applied overnight at 4 °C and then visualized with an Alexa Fluor 568 goat anti-rabbit IgG (H + L) antibody (1:500).

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing

Journal: JARO: Journal of the Association for Research in Otolaryngology

Article Title: Quantitative Analysis of Aquaporin Expression Levels during the Development and Maturation of the Inner Ear

doi: 10.1007/s10162-016-0607-3

Figure Lengend Snippet: Comparison of AQP9 localization to phalloidin (Ph) and myosin 7a (Myo7a) localization in the P21 organ of Corti. The localization of AQP9 in the inner and outer pillar cells were co-localized with phalloidin staining of these cells (a and b, arrows and open arrows, respectively). AQP9 was detected in the Deiter’s cups (a and b, filled arrowheads) beneath the OHCs and in the phalangeal processes (a, open arrowheads) between the OHCs. Bars indicate 20 μm.

Article Snippet: After blocking with 10 % goat-serum in PBS:0.2%Triton-X, rabbit polyclonal anti-Myo7a antibody (1:1000, GeneTex, USA) was applied overnight at 4 °C and then visualized with an Alexa Fluor 568 goat anti-rabbit IgG (H + L) antibody (1:500).

Techniques: Comparison, Staining