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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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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. |
format | Online Article Text |
id | pubmed-8267968 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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