The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1.

Ocular findings in Wt littermates of HLA A29 Tg mice compared to healthy eyes. Fundus photographs were taken at 9–10 weeks of age (A) and SD-OCT at 18 weeks of age (B). Healthy retinas are depicted on the right. (A) Fundus lesions appeared as multiple bright spots in the retina, coalescing to form diffuse lesions or large patches. (The three bright spots visible in the same position on all fundi and the central black spot are camera artifacts.) (B) SD-OCT of eyes are shown in panel A: linear scans of retina showed localized areas of hyperreflectivity and thin retina with loss of inner and outer segment layers, or loss of normal architecture of the inner and outer nuclear layers at focal areas away from the optic nerve; note similarity of these lesions to the ones described by Aleman et al. in mouse Crb1 associated retinal degeneration. (C) Histology – note foci of total photoreceptor atrophy, with loss of outer and inner segments, outer nuclear and outer plexiform layers (left), multiple focal photoreceptor dystrophy without retinal pigment epithelial (RPE) abnormalities (center). (D). Sample gel showing resolution of the rd8 homozygous (244bp), rd8 heterozygous and Wt (220bp) genotypes. GC, ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer; PIS/OS, photoreceptor inner segments/outer segments.

Figure 2.

Ocular finding in AMD-like mice. (A) Fundus photographs from selected mice displaying multiple spots typical of the rd8 phenotype. All mice with the genotype rd8/rd8 had a similar appearance, while Wt/Wt mice were identical to the Ccl2^−/-Cx3Cr1^−/-gld mice shown. (B) Histological finding in selected mice rd8/rd8 and Wt/Wt mice. Note the retinal folding, dysplasia of the nuclear layers, retinal degeneration, and RPE vacuolation. Lesions of this type typically were focal in the eyes examined.

Figure 3.

Genotyping for rd8. (A) Genotyping of vendor mice by PCR for rd8. Bands were resolved by capillary electrophoresis. Mice are from HSD, DCT, CRL, TAC, and JAX. Genotyping for rd8 was performed by PCR. Two representative mice out of 7 obtained from each vendor in 2 separate shipments are shown. (B) Sequencing of Crb1 gene shows a single base deletion in the N substrain at the expected position in the aligned sequences (MegAlign program). N-MF, N substrain sequenced with forward primer; N-MR, N substrain sequenced with reverse primer; J-MF, J substrain sequenced with forward primer; J-MR, J substrain sequenced with reverse primer.

Figure 4.

Vendor C57BL/6N mice, but not C57BL/6J mice, have fundus lesions. (A–E) Fundus photographs of mice whose genotyping is shown in Figure 3A that were obtained from the specified vendors. Shown is one representative mouse out of 7 obtained from each vendor in 2 separate shipments. Note spotty lesions of varying extent in all mice except C57BL/6J. (F) C57BL/10J mice with normal fundi serve as control. (G) Histopathology. Shown are eye sections from affected DCT and CRL C57BL/6N mice compared to normal JAX C57BL/6J mice. Note retinal lesions marked by arrows. Histology of other lesion-positive mice looked essentially identical.