Related Experiment Video
Updated: Mar 28, 2026
The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells
Published on: September 20, 2013
The retinal phenotype of Grk1-/- is compromised by a Crb1 rd8 mutation
Joseph S Pak1, Eun-Jin Lee2, Cheryl Mae Craft1
1Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Departments of Ophthalmology and Cell & Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
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(rd8) ) 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(-/-) ), 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(-/-) with or without the Crb1(rd8) mutation.
Methods:
The mice strains tested were C57BL/6J (B6J), B6N, and Grk1(-/-) on either a B6J (Grk1(-/-) (;B6J)) or B6N background (Grk1(-/-) (;B6N)) and were verified with PCR genotype analysis for Grk1(-/-) and Crb (rd8) . 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(-/-) (;B6N) mice and a significant decrease in the thickness of the ONL in the 3-month-old Grk1(-/-) (;B6N) mice. 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(-/-) (;B6J) mice. In contrast, the B6N and Grk1(-/-) (;B6N) retinas 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(-/-) (;B6J) and Grk1(-/-) (;B6N) retinas versus either the B6J or B6N retinas at 1 and 3 months of age and a small significant difference between the Grk1(-/-) (;B6J) and Grk1(-/-) (;B6N) retinas at 1 month. In addition, glial fibrillary acidic protein (GFAP) expression was enhanced in the Grk1(-/-) (;B6J) and Grk1(-/-) (;B6N) retinas at 1 and 3 months. Occasional sprouting processes of rod bipolar cells were detected in the B6N and Grk1(-/-) (;B6N) retinas, but sprouting was not detected in the B6J or Grk1(-/-) (;B6J) retinas at either age.
Conclusions:
The B6N strain background exhibited abnormal phenotypes in the Grk1(-/-) (;B6N) retina. 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.
Insights
The C57BL/6N mouse strain shows retinal degeneration due to a Crb1 gene mutation. This background influences G-protein receptor kinase 1 knockout phenotypes, impacting photoreceptor health and visual function.
Area of Science:
- Genetics
- Ophthalmology
- Neuroscience
Background:
- Laboratory mouse strains are crucial for genetic research but can harbor spontaneous mutations.
- The C57BL/6N (B6N) strain carries a mutation in the Crumbs homolog 1 (Crb1(rd8)) gene, affecting photoreceptor polarity and causing retinal degeneration.
- This Crb1(rd8) mutation can confound studies using B6N-derived knockout models, such as G-protein receptor kinase 1 knockouts (Grk1(-/-)).
Purpose of the Study:
- To investigate if Grk1(-/-) mice on a B6N background exhibit abnormal retinal phenotypes unrelated to GRK1 function.
- To determine the influence of the Crb1(rd8) mutation on the retinal structure and light-induced damage in Grk1(-/-) mice.
- To assess the impact of the B6N genetic background on the phenotype of Grk1(-/-) mice.
Main Methods:
- Tested mouse strains included C57BL/6J (B6J), B6N, Grk1(-/-) on B6J background (Grk1(-/-);B6J), and Grk1(-/-) on B6N background (Grk1(-/-);B6N).
- Genotypes for Grk1(-/-) and Crb1(rd8) were confirmed using PCR.
- Mice were exposed to light after dark rearing, followed by immunohistochemistry (IHC) and TUNEL assays to evaluate retinal structure, photoreceptor apoptosis, and protein expression (ZO-1, GFAP).
Main Results:
- B6N and Grk1(-/-);B6N mice showed outer nuclear layer (ONL) disorganization and thinning, with fragmented Zona-innervating-1 (ZO-1) staining at the outer limiting membrane (OLM).
- Light exposure significantly increased photoreceptor cell death in Grk1(-/-);B6J and Grk1(-/-);B6N mice compared to controls.
- Enhanced glial fibrillary acidic protein (GFAP) expression and occasional rod bipolar cell sprouting were observed in B6N-derived mice.
Conclusions:
- The C57BL/6N background significantly influences the retinal phenotype of Grk1(-/-) mice, exacerbating light-induced damage and structural abnormalities.
- The Crb1(rd8) mutation present in the B6N strain contributes to these observed phenotypes.
- These findings highlight the importance of considering background strain mutations in genetic knockout studies and their potential impact on visual function.
