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. 2014 Mar 5;9(3):e90342.
doi: 10.1371/journal.pone.0090342. eCollection 2014.

Lrit3 Deficient Mouse (nob6): A Novel Model of Complete Congenital Stationary Night Blindness (cCSNB)

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Free PMC article

Lrit3 Deficient Mouse (nob6): A Novel Model of Complete Congenital Stationary Night Blindness (cCSNB)

Marion Neuillé et al. PLoS One. .
Free PMC article

Abstract

Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Construction of the Lrit3 knock-out allele and genotyping.
(A) Wild-type (wt) Lrit3 allele comprises 4 exons. In the knock-out (ko) construction, exons 3 and 4 were replaced by a selection cassette with only 21 bp of exon 3 still remaining. For genotyping, mLrit3_ex4aF and mLrit3_ex4aR were designed to only amplify the wt allele, whereas mLrit3_ex3F and mLrit3_CasR were designed to only amplify the ko allele. (B) After migration on 2% agarose gel, Lrit3+/+ mice exhibited a single fragment at the expected length of 602 bp, Lrit3nob6/nob6 exhibited a single fragment at the expected length of 377 bp and Lrit3nob6/+ mice exhibited both fragments. Legends: WT: wild-type allele; Mut: mutant allele.
Figure 2
Figure 2. wt and ko Lrit3 cDNAs and RT-PCR.
(A) ko cDNA comprised the remaining 21 bp of exon 3 and only the first 8 bp of the selection cassette which leads to a premature stop codon. mLrit3_RT_ex2F and mLrit3_RT_ex3R were designed to only amplify the wt cDNA whereas mLrit3_RT_ex2F and mLrit3_RT_CasR were designed to only amplify the supposed ko cDNA. (B) After amplification and migration on agarose gel, Lrit3+/+ mice exhibited a single fragment at 539 bp, Lrit3nob6/nob6 mice exhibited a 443 bp fragment and Lrit3nob6/+ mice exhibited both fragments. Legends: WT: wild-type cDNA; Mut: mutant cDNA. (C) Sequence of the ko LRIT3 protein. This 206 amino acid protein are supposed to lack its Ig-like, Serine-rich, fibronectin III, transmembrane and PDZ-binding domains.
Figure 3
Figure 3. Scotopic ERG phenotype.
Dark-adapted ERG series were obtained from representative Lrit3+/+ (black line), Lrit3nob6/+ (blue line) and Lrit3nob6/nob6 (red line) littermates. (A) At 6 weeks of age. The scale indicates 100 ms and 200 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (B) Amplitude of the major components of the dark-adapted ERG with increasing flash intensity at 6 weeks of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (C) Implicit time of the major components of the dark-adapted ERG with increasing flash intensity at 6 weeks of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (D) At 6 months of age. The scale indicates 100 ms and 200 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (E) Amplitude of the major components of the dark-adapted ERG with increasing flash intensity at 6 months of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted. (F) Implicit time of the major components of the dark-adapted ERG with increasing flash intensity at 6 months of age. The b-wave component is absent in Lrit3nob6/nob6 mice and therefore this data is not plotted.
Figure 4
Figure 4. Photopic ERG phenotype.
Cone-mediated ERG series were obtained from representative Lrit3+/+ (black line), Lrit3nob6/+ (blue line) and Lrit3nob6/nob6 (red line) littermates. (A) At 6 weeks of age. The scale indicates 100 ms and 100 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (B) Amplitude of the a-wave with increasing flash intensity at 6 weeks of age. (C) Amplitude of the b-wave with increasing flash intensity at 6 weeks of age. (D) Implicit time of the a-wave with increasing flash intensity at 6 weeks of age. (E) Implicit time of the b-wave with increasing flash intensity at 6 weeks of age. (F) At 6 months of age. The scale indicates 100 ms and 100 µV. Values to the left of the row of waveforms indicate flash intensity in log cd.s/m2. (G) Amplitude of the a-wave with increasing flash intensity at 6 months of age. (H) Amplitude of the b-wave with increasing flash intensity at 6 months of age. (I) Implicit time of the a-wave with increasing flash intensity at 6 months of age. (J) Implicit time of the b-wave with increasing flash intensity at 6 months of age.
Figure 5
Figure 5. Optomotor responses.
The number of head movements per minute was obtained in scotopic conditions with spatial frequencies from 0.063 to 0.75-Wallis statistical test in representative Lrit3+/+ (black box), Lrit3nob6/+ (blue box) and Lrit3nob6/nob6 (red box) littermates. The star indicates a significant test (p<0.05). (A) At 6 weeks of age. (B) At 6 months of age.
Figure 6
Figure 6. Retinal anatomy of Lrit3nob6/nob6 mice.
Retinal sections of representative Lrit3+/+, Lrit3nob6/+ and Lrit3nob6/nob6 littermates were compared by light microscopy at 6 weeks and 6 months of age. Scale bar, 40 µm.
Figure 7
Figure 7. SD-OCT retinal nuclear layers measurements.
(A) ONL, INL and IPL+GCL+NFL thickness were obtained by SD-OCT and compared using Kluskal-Wallis statistical test in representative Lrit3+/+ (black box), Lrit3nob6/+ (blue box) and Lrit3nob6/nob6 (red box) littermates. The star indicates a significant test (p<0.05). (B) At 6 weeks of age. (C) At 6 months of age.

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The project was supported by Agence Nationale de la Recherche [ANR-12-BSVS1-0012-01_GPR179] (CZ), Foundation Voir et Entendre (CZ), Prix Dalloz for “la recherche en ophtalmologie” (CZ), The Fondation pour la Recherche Médicale (FRM) in partnership with the Fondation Roland Bailly (CZ), Ville de Paris and Région Ile de France, LABEX LIFESENSES [reference ANR-10-LABX-65] supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir programme [ANR-11-IDEX-0004-0], Foundation Fighting Blindness center grant [C-CMM-0907-0428-INSERM04]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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