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The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional...

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Autores principales: Koster, Céline, van den Hurk, Koen T., Lewallen, Colby F., Talib, Mays, ten Brink, Jacoline B., Boon, Camiel J. F., Bergen, Arthur A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267968/
https://www.ncbi.nlm.nih.gov/pubmed/34281288
http://dx.doi.org/10.3390/ijms22137234
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author Koster, Céline
van den Hurk, Koen T.
Lewallen, Colby F.
Talib, Mays
ten Brink, Jacoline B.
Boon, Camiel J. F.
Bergen, Arthur A.
author_facet Koster, Céline
van den Hurk, Koen T.
Lewallen, Colby F.
Talib, Mays
ten Brink, Jacoline B.
Boon, Camiel J. F.
Bergen, Arthur A.
author_sort Koster, Céline
collection PubMed
description Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat(−/−) and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat(−/−) animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat(−/−) animals. Lrat(−/−) animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat(−/−) animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat(−/−) rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.
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spelling pubmed-82679682021-07-10 The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa Koster, Céline van den Hurk, Koen T. Lewallen, Colby F. Talib, Mays ten Brink, Jacoline B. Boon, Camiel J. F. Bergen, Arthur A. Int J Mol Sci Article Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat(−/−) and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat(−/−) animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat(−/−) animals. Lrat(−/−) animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat(−/−) animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat(−/−) rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies. MDPI 2021-07-05 /pmc/articles/PMC8267968/ /pubmed/34281288 http://dx.doi.org/10.3390/ijms22137234 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Koster, Céline
van den Hurk, Koen T.
Lewallen, Colby F.
Talib, Mays
ten Brink, Jacoline B.
Boon, Camiel J. F.
Bergen, Arthur A.
The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title_full The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title_fullStr The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title_full_unstemmed The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title_short The Lrat(−/−) Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
title_sort lrat(−/−) rat: crispr/cas9 construction and phenotyping of a new animal model for retinitis pigmentosa
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267968/
https://www.ncbi.nlm.nih.gov/pubmed/34281288
http://dx.doi.org/10.3390/ijms22137234
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