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. 2013 Jul 30;54(7):5111-22.
doi: 10.1167/iovs.13-12336.

Complementation Test of Rpe65 Knockout and tvrm148

Affiliations
Free PMC article

Complementation Test of Rpe65 Knockout and tvrm148

Charles B Wright et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels.

Methods: Normal phase HPLC was used to measure retinoid levels. Rpe65(+/+), tvrm148/+ (T(+/-)), tvrm148/tvrm148 (T(-/-)), RPE65(KO/KO) (Rpe65(-/-)), and Rpe65(T/-) mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein.

Results: The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T(-/-) or Rpe65(-/-) mice. Visual acuity in Rpe65(+/+) and T(+/-) mouse was -0.382 c/d, but 0.037 c/d in T(-/-) mice at postnatal day 210 (P210). ERG response in T(-/-) mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T(-/-) mice was -70% of Rpe65(+/+) by P210. Rpe65 mRNA levels in T(-/-) mice were unchanged, yet 14.5% of Rpe65(+/+) protein levels was detected. Protein size was unchanged.

Conclusions: A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65.

Keywords: RPE65/Rpe65; mutation; visual cycle.

Figures

Figure 1
Figure 1
Rpe65 resides outside the markers D3Mit147 and D3Mit19 on the physical map of chromosome 3. (A) Simplified genetic map of chromosome 3 showing the positions of D3Mit147, D3Mit19, and Rpe65. (B) Ensembl genome browser map of the critical region neighboring the D3Mit147 and D3Mit19 markers based on the GRCm38 build of the mouse genome released in December 2011.
Figure 2
Figure 2
The tvrm148 and Rpe65KO alleles do not complement. Dark-adapted a-wave amplitudes from a flash stimulus of 137 cd·s/m2 from Rpe65+/+ (n = 6), Rpe65−/− (n = 7), T−/− (n = 5), and Rpe65T/− (n = 9) mice at P30 are shown. Rpe65−/−, T−/−, and Rpe65T/− mice have significantly lower a-wave amplitudes than Rpe65+/+ mice, but a-wave amplitudes from Rpe65−/−, T−/−, and Rpe65T/− mice do not differ significantly from one another, showing the two mutant alleles do not complement one another. *P < 0.001 comparing Rpe65+/+ with each of Rpe65−/−, T−/−, and Rpe65T/− mice. Data are presented as mean ± SD.
Figure 3
Figure 3
T−/− mice, but not T+/− mice, have a loss of ERG response. Dark- and light-adapted b-wave amplitudes, respectively, from Rpe65+/+, T+/−, and T−/− mice at P60 (A, B) and P180 (C, D) are shown. *P < 0.05 T−/− compared with Rpe65+/+. **P < 0.001 T−/− compared with Rpe65+/+, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. There was no difference between Rpe65+/+ and T+/− ERG responses. Data are presented as absolute values (mean ± SD).
Figure 4
Figure 4
T−/− mice, but not T+/− mice, lose visual acuity with age. Visual acuities of Rpe65+/+ (solid line); T+/− (dotted line; largely hidden behind the solid back line); and T−/− mice (dashed line) from P30 to P210 are represented on the same graph. **P < 0.001 T−/− compared with Rpe65+/+, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Comparison of T+/− with Rpe65+/+ does not reach statistical significance between P30 and P210, and visual acuity measures overlap through the duration of the study. Data are presented as mean ± SD.
Figure 5
Figure 5
T−/− mice have residual visual function similar to Rpe65−/− mice. Comparison of visual phenotype measured by visual acuity. T−/− mice have a residual visual function similar to Rpe65−/− mice. Visual acuities of Rpe65+/+ (black bar); T−/− (gray bar); and Rpe65−/− (white bar) mice at P30, P120, and P210 are shown on the same graph. Data are presented as mean ± SD. **P < 0.001 compared to wild type. #P < 0.05 T−/− compared with Rpe65−/−. ##P < 0.001 T−/− compared with Rpe65−/−.
Figure 6
Figure 6
T−/− mice have a thinner retina than T+/− or Rpe65+/+ counterparts. Bright-field images of retina cross-sections stained with toluidine blue are shown. T−/− mice experience slight retina thinning but retain normal retina architecture as they age. Representative images of Rpe65+/+, T+/−, and T−/− retina 1 mm superior of the optic nerve at P60 (AC) and P210 (DF) are shown. INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 7
Figure 7
T−/− mouse retina layers progressively thin with age. Quantitative measurements every 500 μm superior and inferior of the optic nerve in Rpe65+/+ (solid line); T+/− (dotted line); and T−/− (dashed line) retina were taken for the number of stacked nuclei in the ONL (A, B); ONL thickness (C, D); IS thickness (E, F); and OS thickness (G, H) at P60 (Rpe65+/+ n = 11, T+/− n = 8, T−/− n = 12) and P210 (Rpe65+/+ n = 7, T+/− n = 6, T−/− n = 14). *P < 0.05 T−/− compared with Rpe65+/+. **P < 0.001 T−/− compared with Rpe65+/+. +P < 0.05 Rpe65+/+ compared with T+/−, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Data are presented as mean ± SD.
Figure 8
Figure 8
T−/− mice have significant cone nuclei loss at P60 and P210. Cone nuclei were counted at distances of 0.25, 1, and 2 mm superior and inferior of the optic nerve in a 200-μm segment of the retina at (A) P60 and (B) P210. **P < 0.001 Rpe65+/+ compared with T−/−, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Data are presented as mean ± SD.
Figure 9
Figure 9
TEM shows lipid droplets indicative of retinyl ester accumulation in the RPE of T+/− and T−/− mice. TEM was performed on sections of RPE 1000 μm superior to the optic nerve in Rpe65+/+, T+/−, and T−/− mice at P60 and P210. (AC) TEM images of RPE from Rpe65+/+, T+/−, and T−/− mice at P60, respectively, at ×2900 magnification are shown. (DF) TEM images of Rpe65+/+, T+/−, and T−/− mice at P210, respectively, at ×2900 magnification are shown. (GI) TEM images of Rpe65+/+, T+/−, and T−/− mice at P210, respectively, at ×10,000 magnification are shown. N, nucleus; L, lipid droplet.
Figure 10
Figure 10
T−/− mice express Rpe65 mRNA at the same level as Rpe65+/+ mice under steady-state conditions. Whole cell RNA from P60 Rpe65+/+ and T−/− RPE/choroid extracts was harvested for qRT-PCR. Steady-state Rpe65 mRNA levels in Rpe65+/+ mice (n = 4) and T−/− mice (n = 5) show no significant difference (P = 0.617).
Figure 11
Figure 11
Immunoblots and densitometry for RPE65 in Rpe65+/+, T+/−, and T−/− mice show mutant mice have reduced protein levels. Protein extracts harvested from P60 Rpe65+/+ and T−/− RPE. (A) Immunoblotting for RPE65 in Rpe65+/+, T+/−, and T−/− protein extracts showed a decrease in RPE65 levels in T+/− and T−/− mice but no change in protein size. Gapdh is shown as a loading control. (B) Densitometry of immunoblots showed 60 ± 4.7% Rpe65+/+ levels of RPE65 protein in T+/− mice and 14.5 ± 1.5% protein levels in T−/− mice. Data are presented as percentage change from Rpe65+/+ (mean ± SD).

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