June 2012
Volume 53, Issue 7
Letters to the Editor  |   June 2012
Retinal Lesions and Other Potential Confounders of Ocular Research in Inbred Mice
Author Affiliations
  • Kathleen R. Pritchett-Corning
    Research Models and Services, Charles River, Wilmington, Massachusetts; and
  • Charles B. Clifford
    Research Models and Services, Charles River, Wilmington, Massachusetts; and
  • Bruce J. Elder
    Research Models and Services, Charles River, Wilmington, Massachusetts; and
  • Mark Vezina
    Preclinical Services, Charles River, Montreal, Quebec, Canada.
    E-mail: kathleen.pritchett@crl.com
Investigative Ophthalmology & Visual Science June 2012, Vol.53, 3764-3765. doi:10.1167/iovs.12-10056
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      Kathleen R. Pritchett-Corning, Charles B. Clifford, Bruce J. Elder, Mark Vezina; Retinal Lesions and Other Potential Confounders of Ocular Research in Inbred Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(7):3764-3765. doi: 10.1167/iovs.12-10056.

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      © ARVO (1962-2015); The Authors (2016-present)

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We read with interest a study recently published describing retinal lesions seen in C57BL6/N (B6N) mice and attributing the phenotype to a mutation, called rd8, in a gene, Crb1. 1 It appears to be fixed (present at 100% frequency) in all substrains of B6N mice, but is not present in C57BL/6J (B6J) mice. This mutation also appears to be fixed in B6N derived embryonic stem cell lines, which are being used by knockout mouse consortia, such as those in IMKC. 2,3 It is important to note that this mutation is not new, nor is this the first published report of it. 4,5 While the current paper speculates that there are modifier genes present in the B6N background that result in a more extensive phenotype in the B6N background when compared with B6J, other groups studying the mutation have noted that normal retinal function is largely retained by mice with this mutation. 6 Aleman et al. go on to conclude that mice with the mutation are probably relatively normally sighted and therefore not an ideal model of human ocular diseases involving the homologous gene. 6  
Mutations happen in every biological system and are not preventable. The great majority of mutations are recessive and may not result in a change in phenotype visible to caretakers even when a mutation becomes fixed in an inbred population. Mutations of the Snca gene in B6JOlaHsd 7 and the Nnt gene 8 in B6J mice are classic examples of this. In fact, it is fixed mutations in the various inbred lines that provide their characteristic traits. For example, the Crb1rd8 mutation must have occurred before the NIH founder lines were distributed to the major vendors (i.e., prior to the 1970s). As a result, if any current C57BL/6N lines lacked the mutation, it could be evidence of cross-breeding with another line. 
Many of these common mutations may have no currently described functional effect even if they are phenotypically evident (such as some mutations of coat color), while others affect disease resistance, or cause lesions, such as dystrophic calcification, microphthalmia, anophthalmia, or hydrocephalus. Often, scientists choose a C57BL/6 background because it is commonly available and widely used by researchers in many fields. It is not always the best choice, however. For example, in addition to Crb1rd8 present in the B6N substrains, all B6 mice have several inherited ocular phenotypes with variable penetrance, so why choose an animal prone to micro- and anophthalmia as well as corneal opacities and anterior polar cataracts for vision research? 911 In addition, many investigators are unaware that variance in substrains of B6 mice between experimental and control mice may significantly confound research results. 12 Working with knockout mouse models may present further challenges, as many models were created using embryonic stem cell lines of 129 origin, which have a suite of ubiquitous mutations of their own. 1315 Backcrossing animals originating from 129 embryonic stem cells onto a desired background can also present risks, as flanking DNA of 129 origin always remains linked with the gene of interest. 16  
Below is a much-abbreviated list of other genes with documented mutant alleles known to be in certain strains of mice that could potentially affect research. Although each individual mutant or variant allele may not directly affect ocular research, they might interact with other genes and influence ocular phenotypes. 
Pdeb6rd1 is the classical rd1 retinal degeneration gene found in many inbred mice. There are at least 16 other retinal degeneration mutations described, not including separate mutations that affect the cornea, lens, or other ocular structures. 5 NntC57BL6/J is involved in glucose metabolism and liver function 8 and fixed in the B6J population, but not in the B6N. Mutations in the Snca gene may affect the study of Parkinson's disease. A mutated allele was first described in a subpopulation of B6J mice maintained at an European facility of Harlan. 7 Caspases such as Casp4 play roles in inflammation, necrosis, and apoptosis and mutations in this gene have been found in 129 origin ES cells. 13,14 Disc1del is a mutation in a gene associated with schizophrenia in humans and present in all sublines within the 129 family of mice. 15 Hc0 affects the complement cascade and animals with this allele are deficient in C5. A mutant allele, Tlr4lps-d , found in C3H/HeJ mice confers resistance to endotoxin. 17,18 Abcc6 is the gene responsible for dystrophic calcinosis in mice and a variant allele is present in a number of inbred strains, notably BALB/c, BALB/cBy, and DBA/2. 19 Cdh23ahl is a mutation present in at least 10 commonly used strains of mice resulting in age-related hearing loss. 20  
Investigators should be aware that all inbred mice inevitably carry mutations relevant to some areas of research, and that all observed phenotypic changes initially attributed to genetic manipulation must be carefully considered in the context of background genotype, environment, and microbiome. 21,22 Research mice are complex biological systems that will have inherent variation and inexorable change over time. 23 Investigators should also be cognizant of how the environment can influence the expression of genes (epigenetics) and how this could potentially affect ocular development. 24  
Producers of laboratory rodents cannot know all the mutations fixed or present in inbred or outbred populations. We rely significantly on the scientific community to communicate the discovery of new mutations or alterations in phenotypic expression. When a new mutation is identified, laboratory rodent producers appreciate timely notification and a measured response from the scientific community. Scientists also have the responsibility to thoroughly investigate the “normal” background characteristics of the strains in which they plan to work. This will allow potentially induced phenotypes or lesions to be distinguished from strain-related phenotypes that may have been previously described. 2528  
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Pettitt SJ Liang Q Rairdan XY Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat Methods . 2009;6:493–495. [CrossRef] [PubMed]
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Bourdi M Davies JS Pohl LR . Mispairing C57BL/6 substrains of genetically engineered mice and wild-type controls can lead to confounding results as it did in studies of JNK2 in acetaminophen and concanavalin A liver injury. Chem Res Toxicol . 2011;24:794–796. [CrossRef] [PubMed]
Kenneth NS Younger JM Hughes ED An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1. Biochem J . 2012;443:355–359. [CrossRef] [PubMed]
Kayagaki N Warming S Lamkanfi M Non-canonical inflammasome activation targets caspase-11. Nature . 2011;479:117–121. [CrossRef] [PubMed]
Clapcote SJ Roder JC . Deletion polymorphism of Disc1 is common to all 129 mouse substrains: implications for gene-targeting studies of brain function. Genetics . 2006;173:2407–2410. [CrossRef] [PubMed]
Boross P Arandhara VL Martin-Ramirez J The inhibiting Fc receptor for IgG, FcgammaRIIB, is a modifier of autoimmune susceptibility. J Immunol . 2011;187:1304–1313. [CrossRef] [PubMed]
Dumont F Barrois R . Electrokinetic properties of splenic lymphocytes from the low-lipopolysaccharide responder C3H/HeJ mice. Folia Biol (Praha) . 1976;12:145–150. [PubMed]
Poltorak A He X Smirnova I Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science . 1998;282:2085–2088. [CrossRef] [PubMed]
Meng H Vera I Che N Identification of Abcc6 as the major causal gene for dystrophic cardiac calcification in mice through integrative genomics. Proc Natl Acad Sci U S A . 2007;104:4530–4535. [CrossRef] [PubMed]
Johnson KR Zheng QY Erway LC . A major gene affecting age-related hearing loss is common to at least ten inbred strains of mice. Genomics . 2000;70:171–180. [CrossRef] [PubMed]
Sellers RS Clifford CB Treuting PM Brayton C . Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice. Vet Pathol . 2012;49:32–43. [CrossRef] [PubMed]
Treuting PM Clifford CB Sellers RS Brayton CF . Of mice and microflora: considerations for genetically engineered mice. Vet Pathol . 2012;49:44–63. [CrossRef] [PubMed]
Sellers RS . The gene or not the gene—that is the question: understanding the genetically engineered mouse phenotype. Vet Pathol . 2012;49:5–15. [CrossRef] [PubMed]
Cvekl A Mitton KP . Epigenetic regulatory mechanisms in vertebrate eye development and disease. Heredity (Edinb) . 2010;105:135–151. [CrossRef] [PubMed]
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Brayton C . Spontaneous diseases in commonly used mouse strains/stocks . Available at: http://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/09s1BraytonMouseDzOutline75p.pdf. Accessed April 12, 2012.
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