TY - JOUR
T1 - The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa
AU - Koster, C. line
AU - van den Hurk, Koen T.
AU - Lewallen, Colby F.
AU - Talib, Mays
AU - ten Brink, Jacoline B.
AU - Boon, Camiel J. F.
AU - Bergen, Arthur A.
N1 - Funding Information: Funding: This project was (in part) funded by the following grants: Stichting Uitzicht (UZ 2016–21 including the Stichting MD Nederland, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Retina Fonds Nederland, Oogfonds), and the the Rotterdamse Stichting Blindenbelangen (#B20160043), Stichting Blindenhulp, Stichting voor Ooglijders and Stichting Lijf en Leven (#42) (all to A.A.B). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/7/1
Y1 - 2021/7/1
N2 - 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.
AB - 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.
KW - Lecithin retinol acyltransferase (LRAT)
KW - Pigmented rat model
KW - Retinal degeneration
KW - Retinal dystrophy
KW - Retinal pigment epithelium (RPE)
KW - Retinitis pigmentosa
UR - http://www.scopus.com/inward/record.url?scp=85111857880&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/ijms22137234
DO - https://doi.org/10.3390/ijms22137234
M3 - Article
C2 - 34281288
SN - 1661-6596
VL - 22
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 13
M1 - 7234
ER -