title: The retinal phenotype of Grk1-/- is compromised by a Crb1 rd8 mutation.
abstract: OBJECTIVE
  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.
authors.affiliation: Mary 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.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Pak
authors.affiliation: Mary 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 ; Department of Biomedical Engineering, University of Southern California Viterbi School of Engineering, CA.
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Mary 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.
authors.forename: Cheryl Mae
authors.initials: CM
authors.lastname: Craft
mesh_terms: D000367:Age Factors; D000818:Animals; D017209:Apoptosis; D003624:Darkness; D005260:Female; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000172:deficiency / Q000235:genetics*; D005786:Gene Expression Regulation; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010775:Photic Stimulation; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction; D013045:Species Specificity; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C453730:Crb1 protein, mouse; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C528900:glial fibrillary astrocytic protein, mouse; D051606:G-Protein-Coupled Receptor Kinase 1; C496762:Grk1 protein, mouse