The retinal phenotype of Grk1 super(-)/-is compromised by a Crb1 super(r)d8mutation

Purpose: Well-established laboratory mouse lines are important in creating genetically engineered knockout mouse models; however, these routinely used inbred strains are prone to spontaneous and deleterious mutations. One of these strains, the commonly used C57BL/6N (B6N), was discovered to carry a...

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Bibliographic Details
Published inMolecular vision Vol. 21; pp. 1281 - 1294
Main Authors Pak, Joseph S, Lee, Eun-Jin, Craft, Cheryl Mae
Format Journal Article
LanguageEnglish
Published 01.01.2015
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Summary:Purpose: Well-established laboratory mouse lines are important in creating genetically engineered knockout mouse models; however, these routinely used inbred strains are prone to spontaneous and deleterious mutations. One of these strains, the commonly used C57BL/6N (B6N), was discovered to carry a point mutation in the Crumbs homolog 1(Crb1 super(r)d8 gene, which codes for a developmental protein involved in tight junction formation at the outer limiting membrane (OLM). This mutation disrupts photoreceptor polarity and leads to retinal degeneration. It was hypothesized that the G-protein receptor kinase 1 knockouts (Grk1 super(-)/- , which were based on the B6N strain, would exhibit abnormal morphological phenotypes in their offspring not related to GRK1's major phosphorylation function. The hypothesis was tested by examining Grk1 super(-)/-with or without the Crb1 super(r)d8mutation. Methods: The mice strains tested were C57BL/6J (B6J), B6N, and Grk1 super(-)/-on either a B6J (Grk1 super(-)/- super(; )B6J or B6N background (Grk1 super(-)/- super(; )B6N and were verified with PCR genotype analysis for Grk1 super(-)/-and Crb super(r)d8 The mice were bred and raised in complete darkness until 1 or 3 months of age and then exposed to 1,000 lux light for 24 h, followed by processing for immunohistochemistry (IHC) analysis on the retinal structure to investigate the morphological effects of light exposure. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to detect photoreceptor apoptosis. Results: The microanatomy of the retinal sections revealed disorganization of the outer nuclear layer (ONL) in the B6N and Grk1 super(-)/- super(; )B6Nmice and a significant decrease in the thickness of the ONL in the 3-month-old Grk1 super(-)/- super(; )B6Nmice. The adherens-junction-associated protein, Zona occludens-1 (ZO-1), formed a continuous line at the OLM in the 1- and 3-month-old control B6J and Grk1 super(-)/- super(; )B6Jmice. In contrast, the B6N and Grk1 super(-)/- super(; )B6Nretinas showed discontinuous and fragmented staining for ZO-1 at the OLM at both ages. After the mice were exposed to light, TUNEL analysis showed a significant increase in photoreceptor cell death in the Grk1 super(-)/- super(; )B6Jand Grk1 super(-)/- super(; )B6Nretinas versus either the B6J or B6N retinas at 1 and 3 months of age and a small significant difference between the Grk1 super(-)/- super(; )B6Jand Grk1 super(-)/- super(; )B6Nretinas at 1 month. In addition, glial fibrillary acidic protein (GFAP) expression was enhanced in the Grk1 super(-)/- super(; )B6Jand Grk1 super(-)/- super(; )B6Nretinas at 1 and 3 months. Occasional sprouting processes of rod bipolar cells were detected in the B6N and Grk1 super(-)/- super(; )B6Nretinas, but sprouting was not detected in the B6J or Grk1 super(-)/- super(; )B6Jretinas at either age. Conclusions: The B6N strain background exhibited abnormal phenotypes in the Grk1 super(-)/- super(; )B6Nretina. This study demonstrates that the B6N background can influence the phenotype of a genetic mouse knockout and introduces potential visual functional consequences of the Crb1 mutation.
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ISSN:1090-0535
1090-0535