The<i>Rd8</i>Mutation of the<i>Crb1</i>Gene Is Present in Vendor Lines of C57BL/6N Mice and Embryonic Stem Cells, and Confounds Ocular Induced Mutant Phenotypes

TLDR

The study aimed to determine how widespread the rd8 mutation is in C57BL/6N mice, vendor lines, and embryonic stem cells used for ocular disease models. The authors screened mice and ES cells for the rd8 mutation using PCR, confirmatory DNA sequencing, and assessed ocular lesions by fundus examination and histopathology. The rd8 mutation was found in all C57BL/6N substrains and in C57BL/6N‑derived embryonic stem cells, and ocular lesions in induced mutant lines were entirely attributable to this mutation, underscoring the need for routine screening in C57BL/6N‑based studies.

Abstract

We noted an unexpected inheritance pattern of lesions in several strains of gene-manipulated mice with ocular phenotypes. The lesions, which appeared at various stages of backcross to C57BL/6, bore resemblance to the rd8 retinal degeneration phenotype. We set out to examine the prevalence of this mutation in induced mutant mouse lines, vendor C57BL/6 mice and in widely used embryonic stem cells.Ocular lesions were evaluated by fundus examination and histopathology. Detection of the rd8 mutation at the genetic level was performed by PCR with appropriate primers. Data were confirmed by DNA sequencing in selected cases.Analysis of several induced mutant mouse lines with ocular disease phenotypes revealed that the disease was associated 100% with the presence of the rd8 mutation in the Crb1 gene rather than with the gene of interest. DNA analysis of C57BL/6 mice from common commercial vendors demonstrated the presence of the rd8 mutation in homozygous form in all C57BL/6N substrains, but not in the C57BL/6J substrain. A series of commercially available embryonic stem cells of C57BL/6N origin and C57BL/6N mouse lines used to generate ES cells also contained the rd8 mutation. Affected mice displayed ocular lesions typical of rd8, which were detectable by funduscopy and histopathology as early as 6 weeks of age.These findings identify the presence of the rd8 mutation in the C57BL/6N mouse substrain used widely to produce transgenic and knockout mice. The results have grave implications for the vision research community who develop mouse lines to study eye disease, as presence of rd8 can produce significant disease phenotypes unrelated to the gene or genes of interest. It is suggested that researchers screen for rd8 if their mouse lines were generated on the C57BL/6N background, bear resemblance to the rd8 phenotype, or are of indeterminate origin.