key: cord-0017740-sqvv556u
authors: Mekada, Kazuyuki; Yoshiki, Atsushi
title: Substrains matter in phenotyping of C57BL/6 mice
date: 2021-01-14
journal: Exp Anim
DOI: 10.1538/expanim.20-0158
sha: c2ec217faaf117a4f8aa2e89548be64642fce3d2
doc_id: 17740
cord_uid: sqvv556u

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.

| Exp. Anim. 2021; 70(2): [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] C57BL/6 substrain. We interviewed several scientists who either developed or deposited these strains and found that they had purchased and used the mice without knowing about the substrain distinctions or had not kept detailed breeding records. similar occurrences have been reported elsewhere. over 70% of the genetically engineered strains generated at German and austrian institutions had a mixed genetic background [14] . a survey of Finnish institutions revealed that 39.5% of researchers did not consider the importance of differences among substrains and that 26% did not know which C57BL/6 substrain they had used [15] . Furthermore, 58% of the studies that were published in Diabetes during 2010-2014 and involved genetically modified mice provided incomplete descriptions of the background strains [16] . Despite the reported non-negligible phenotypic differences among C57BL/6 substrains, there seems to be little awareness of the importance of substrain selection within the research community.

To date, dozens of papers have reported various phenotypic differences among C57BL/6 substrains ( Table  1 ). This information is classified according to the phenotype terms defined in the Mammalian Phenotype Ontology [116] and includes descriptions of phenotypes or phenotyping tests, the names of tested C57BL/6 substrains, and relevant references. Moreover, several of these phenotypic differences have been analyzed with the aim of identifying causative genes or genomic regions, as described below (summarized in Table 2 ).

The C57BL/6J strain is missing the exon that encodes the nicotinamide nucleotide transhydrogenase (Nnt) gene, which plays an important role in glucose homeostasis and insulin secretion, and therefore has abnormal glucose tolerance and impaired insulin secretion [117, 118, 126] . a study using mitogen-activated protein kinase 9 (Mapk9) knockout mice demonstrated that susceptibility to acetaminophen-induced liver injury was influenced by the presence or absence of the Nnt gene defect carried by C57BL/6 mice [127] . The Nnt gene mutation in the C57BL/6J strain is a prominent phenotype-associated mutation in C57BL/6 substrains that affects protein expression in the mitochondria of many tissues and consequently many different metabolic traits [62, 72, 78, 82, 88, 106, 128] . The Nnt gene deletion status differs among C57BL/6J substrains, and the Nnt gene defect likely originated in C57BL/6J mice at the Jackson Laboratory after 1976 (Fig. 3 ). C57BL/6Jolahsd mice are known to lack a 365 kb genomic region that includes the synuclein, alpha (Snca) and the nearby multimerin 1 (Mmrn1) gene [119, 120] . sNCa aggregates in the nervous system of individuals with Parkinson's disease [129] . Loss of the Snca gene in C57BL/6Jolahsd mice does not appear to contribute to prion disease-mediated synaptotoxicity or neurodegeneration but may affect motor neuron degeneration [130] [131] [132] [133] . in addition, the C57BL/6Jolahsd strain has a low trabecular bone mass, which has been shown to be associated with the lack of Snca and Mmrn1 genes, suggesting a role for these genes in bone metabolism [110] . Deletion of this genomic region is specific to the C57BL/6Jolahsd strain; the mutation has not been identified in C57BL/6JRccHsd, C57BL/6J, C57BL/6NCrl, and C57BL/6Nhsd strains [119] . Notably, we confirmed that the genotype is normal in other C57BL/6J and C57BL/6N substrains available in Japan (supplementary Fig. 1 ). Furthermore, partial deletion of a chromosomal region was recently observed in C57BL/6JBomTac mice. in this strain, approximately 40 Mb of the long arm of the Y chromosome is deleted, and an increased rate of sperm morphological abnormalities, unbalanced sex ratio, and deregulation of several transcripts expressed in the testes have also been observed [70, 125] .

Similarly, genetic mutations affecting physiological functions have been reported in C57BL/6N substrains. a nonsense mutation (retinal degeneration 8, rd8) that converts the amino acid codon of the crumbs family member 1, photoreceptor morphogenesis associated (Crb1) gene into a termination codon occurs in C57BL/6N substrains [121, 122] . Affected mice present with the typical lesions of rd8, which are detected by fundoscopy and histopathology at as early as 6 weeks of age. although this mutation is seen in C57BL/6N substrains, C57BL/6J substrains have the wild-type genotype [121] .

C57BL/6Nhsd mice have a copy-number variant in the dedicator of cytokinesis 2 (Dock2) gene, which has been shown to affect B cell signaling and immune tolerance, but this is not found in other C57BL/6 substrains [123, 134] . The dysfunction of this gene has likely contributed to the negative outcomes of experiments using the sialic acid acetylesterase (Siae) knockout mice with an unclear C57BL/6 origin. although the Siae gene was initially thought to be involved in B cell development and signal transduction, it was found that congenic mice generated by backcrossing to the C57BL6J strain did not reproduce the same negative outcomes. after much research, the cause was identified as a mutation in the Dock2 gene of the C57BL/6Nhsd strain [134] . [17] alcohol preference C57BL/6J (JaX), C57BL/6NCrsim (simonsen Lab) [18] C57BL/6J (JaX), C57BL/6NCrl (CRL) [19] C57BL/6J (JaX), C57BL/6J (Univ. of albeta) [20] C57BL/6J (JaX), C57BL/6NCrl (CRL) [21] alcohol preference and innate immune response C57BL/6J (JaX), C57BL/6NJ (JaX) [22] Behavioral response to amphetamine C57BL/6J (JaX), C57BL/6By (JaX) [23] Conditioned place preference C57BL/6J (JaX), C57BL/6NCrl (CRL) [24] Corticosterone sensitivity C57BL/6J (JaX), C57BL/6NCrl (CRL) [25] degree of cuprizone-induced binge-like eating reduction C57BL/6J (JaX), C57BL/6NJ (JaX) [26] degree of naloxone-induced conditioned place aversion C57BL/6J (JaX), C57BL/6NJ (JaX) [27] Eight-arm radial maze, elevated plus maze, open field, rotarod, Porsolt forced swim, prepulse inhibition, wire hang C57BL/6J (JaX), C57BL/6NCrlCrlj (CRL Japan), C57BL/6NCrslc (Japan sLC) [28] elevated plus maze, fear conditioning, light-dark box, prepulse inhibition, open field, social approach, acoustic startle C57BL/6J (JaX), C57BL/6JRcchsd (envigo), C57BL/6JRj (Janvier Labs), C57BL/6NCrl (CRL), C57BL/6Nhsd (envigo), C57BL/6NRj (Janvier Labs) [15] elevated plus maze, hole board, light-dark box C57BL/6Jolahsd (envigo), C57BL/6NCrl (CRL) [29] Fear conditioning C57BL/6Jico (CRL), C57BL/6NCrl (CRL) [30] C57BL/6J (hsd) (envigo), C57BL/6NCrl (CRL) [31] C57BL/6J (JaX), C57BL/6Jolahsd (envigo), C57BL/6NCrl (CRL) [32] C57BL/6Jolahsd (envigo), C57BL/6NCrl (CRL) [33] C57BL/6Jolahsd (envigo), C57BL/6NCrlBR (CRL) [34] Fear conditioning, hot plate, rotarod, tail withdrawal C57BL/6J (JaX), C57BL/6NCrl (CRL), C57BL/6Nhsd (envigo), C57BL/6NTac (TaC) [35] Fear conditioning, Morris water maze, open field, rotarod C57BL/6J (JaX), C57BL/6NJ (JaX) [36] Food consumption in conditioned site preference C57BL/6J (JaX), C57BL/6NJ (JaX) [37] high-fat diet-induced mechanical sensitivity C57BL/6J (JaX), C57BL/6NCrl (CRL), C57BL/6NJ (JaX) [38] Light-dark box C57BL/6J (envigo), C57BL/6ChR (CRL) [39] Locomotor activity and anxiety-like behavior during the postpartum period C57BL/6J (JaX), C57BL/6JJcl (CLea Japan) [40] Locomotor response to cocaine C57BL/6J (JaX), C57BL/6N (NCi) [41] Metabolic and behavioral response to shortened 21-h day and high-fat diet C57BL/6J (JaX), C57BL/6NCrl (CRL) [42] Morris water maze C57BL/6J (JaX), C57BL/6NTac (TaC) [43] Open field C57BL/6J (JaX), C57BL/6NTac (TaC) [44] Open field, rotarod C57BL/6JBomTac (TaC), C57BL/6NCrljori (orient Bio), C57BL/6NTacsam (samtako Bio) [45] Prepulse inhibition C57BL/6J (JaX), C57BL/6Nhsd (envigo) [46] Response to formalin-induced pain C57BL/6J (JaX), C57BL/6NCrl (CRL) [47] Response to circadian disruption and wheel-running access C57BL/6J (JaX), C57BL/6NCrl (CRL) [48] Response to neuropathic pain C57BL/6NCrl (CRL), C57BL/6NTac (TaC) [49] sensitivity to the convulsant pilocarpine C57BL/6J (JaX), C57BL/6NJ (JaX) [50] C57BL/6J (JaX), C57BL/6Jolahsd (envigo), C57BL/6NCrl (CRL), C57BL/6Nhsd (envigo) [51] sensitivity to nicotine C57BL/6J (JaX), C57BL/6NCrl (CRL) [52] sensitivity to thermal nociception C57BL/6J (JaX), C57BL/6NJ (JaX) [53] susceptibility to cocaine-induced seizure C57BL/6J (JaX), C57BL/6ByJ (JaX) [54] susceptibility to pilocarpine-induced status epilepticus C57BL/6JJcl (CLea Japan), C57BL/6NJcl (CLea Japan) [55] Tail suspension C57BL/6J (JaX), C57BL/6Nhsd (envigo) [56] | Exp. Anim. 2021; 70(2): 145-160

Phenotype terms descriptions and/or tests C57BL/6 substrains tested Publication

Cardiac effects of tribromoethanol and ketamine-midazolam C57BL/6J (JaX), C57BL/6NCrl (CRL) [57] Cardiac functional and metabolic flux parameters C57BL/6J (JaX), C57BL/6NCrl (CRL) [58] echocardiographic and electrocardiographic values C57BL/6J (hZM), C57BL/6N (hZM) [59] incidence of cardiac rupture after myocardial infarction C57BL/6J (JaX), C57BL/6J (hsd) (envigo) [60] incidence of dilated cardiomyopathy C57BL/6J (JaX), C57BL/6NTac (TaC) [61] high-fat diet-induced vascular superoxide C57BL/6J (JaX), C57BL/6NJ (JaX) [62] Response to ang ii-dependent left ventricle remodeling C57BL/6J (JaX), C57BL/6NJ (JaX) [63] Response to left ventricular pressure overload C57BL/6J (JaX), C57BL/6NCrl (CRL), C57BL/6NTac (TaC) [64] Response to neonatal hypoxia/ischemia C57BL/6J (JaX), C57BL/6NCrl (CRL) [65] Response to transverse aortic constriction stimulation C57BL/6J (JaX), C57BL/6NTac (TaC) [66] salt sensitivity of blood pressure C57BL/6J (JaX), C57BL/6NTac (TaC) [67] systolic blood pressure level C57BL/6J (JaX), C57BL/6NJ (JaX) [68] Vulnerability to neonatal hypoxia-ischemia C57BL/6J (saarland Univ.), C57BL/6N (saarland Univ.) [69] Cellular phenotype abnormal sperm head morphology C57BL/6JBomTac (TaC), C57BL/6NTac (TaC) [70] hydrogen peroxide-producing capacities of liver mitochondria C57BL/6J (JaX), C57BL/6NCrl (CRL) [71] Mitochondrial redox abnormalities C57BL/6J (JaX), C57BL/6JUnib (CeMiB) [72] embryonic phenotype incidence of fetal alcohol syndrome C57BL/6J (JaX), C57BL/6NCrl (CRL) [73] Patterns of alcohol-induced facial dysmorphology C57BL/6J (JaX), C57BL/6Nhsd (envigo) [74] susceptibility to Porphyromonas gingivalis C57BL/6J (JaX), C57BL/6NJ (JaX) [75] endocrine/exocrine gland phenotype susceptibility to cerulein-induced chronic pancreatitis C57BL/6J (JaX), C57BL/6Nhsd (envigo) [76] hearing/vestibular/ear phenotype susceptibility to aminoglycoside-induced ototoxicity C57BL/6J (JaX), C57BL/6Nhsd (envigo) [77] hematopoietic system phenotype sensitivity of hematopoietic stem cells to oxidative stress C57BL/6J (JaX), C57BL/6NCrl (CRL) [78] homeostasis/metabolic phenotype Clinical chemistry parameters C57BL/6J (JaX), C57BL/6NTac (TaC) [79] Effects of oats on plasma cholesterol and lipoproteins C57BL/6JBomTac (TaC), C57BL/6NCrl (CRL) [80] hematological and iron-related parameters C57BL/6J (JaX), C57BL/6NCrl (CRL) [81] insulin secretory response to high-fat diet C57BL/6J (JaX), C57BL/6J (Wehi), C57BL/6NCrl (CRL), C57BL/6Nhsd (envigo), C57BL/6NJ (JaX), C57BL/6NTac (TaC) [82] Response to intermittent hypoxia C57BL/6J (JaX), C57BL/6NCrl (CRL) [83] Response to diet-induced obesity C57BL/6JRj (Janvier Labs), C57BL/6NTac (TaC) [84] C57BL/6JRj (Janvier Labs), C57BL/6NTac (TaC) [85] C57BL/6J (JaX), C57BL/6NJ (JaX) [86] Response to glucose-stimulated insulin secretion C57BL/6J (JaX), C57BL/6NCrl (CRL) [87] Response to high-fat diet C57BL/6J (JaX), C57BL/6NJ (JaX) [88] C57BL/6J (JaX), C57BL/6JBomTac (TaC), C57BL/6JRj (Janvier Labs) [89] skeletal and metabolic responses to high-fat diet C57BL/6J (JaX), C57BL/6NCrl (CRL) [90] immune system phenotype

Neutrophil recruitment in response to inflammatory stimuli C57BL/6J (JaX), C57BL/6N (NCi) [91] Non-ecotropic eRV expression level C57BL/6J (JaX), C57BL/6NJ (JaX) [92] Susceptibility to influenza A virus C57BL/6J (JaX), C57BL/6NCrl (CRL) [93] susceptibility to Listeria monocytogenes C57BL/6J (JaX), C57BL/6ByJ (JaX) [94] integument phenotype Sensitivity to Aldara™-induced psoriasiform dermatitis C57BL/6JRj (Janvier Labs), C57BL/6NRj (Janvier Labs) [95] Liver/biliary system phenotype susceptibility to acetaminophen-induced liver injury C57BL/6J (JaX), C57BL/6NCr (Nih) [96] susceptibility to CCl 4 -and high-fat diet-induced nonalcoholic steatohepatitis C57BL/6J (JaX), C57BL/6NCrslc (Japan sLC) [97] Neoplasm phenotype incidence of 1,2-dimethylhydrazine-induced colorectal tumors C57BL/6J (JaX), C57BL/6N (Nih), C57BL/6ha (health Research inc.) [98] susceptibility to ehrlich ascites carcinoma C57BL/6J (andreevka), C57BL/6N (Pushchino) [99] Other Phenotypic Differences Among C57BL/6 Substrains in addition to the abovementioned cases in which the causative genes were identified, phenotypic differences have been reported among the substrains used to generate the C57BL/6 congenic strain. Greater improvement in terms of seizure severity and viability was achieved in dravet syndrome model mice with knockout of the sodium channel, voltage-gated, type i, alpha (Scn1a) gene generated on a C57BL/6NJ versus C57BL/6J background [135] . Obvious phenotypic differences have been demonstrated between C57BL/6J and C57BL/6N substrains, as shown in studies on knockout of the coagulation factor Viii (F8) and fatty acid binding protein 1, liver (Fabp1) genes, suggesting the presence of genetic factors that differentially modify phenotypes between the substrains [136, 137] . it has also been demonstrated that tumor cell rejection in eµ-TCL1 transgenic mice, a model of chronic lymphocytic leukemia, is associated with differences between the C57BL/6 substrains, suggesting that acute immune rejection may be mediated by antigens that differ between C57BL/6J and C57BL/6N mice [138] . in addition, C57BL/6J-XY Pos congenic mice, which have a Y chromosome derived from Mus musculus poschiavinus on a C57BL/6J genetic background, form ovotestes or ovaries [139, 140] . Changing from a C57BL/6J to C57BL/6N background increases the likelihood of testis-forming individuals, suggesting that genetic differences involved in the regulation of the sex determining region of Chr Y (Sry) and the sRY (sex determining region Y)-box 9 (Sox9) gene expression, which play an important role in testicular differentiation, exist between the C57BL/6J and N substrains [141, 142] . Ectopic distribution of cerebrospinal fluid contacting neurons C57BL/6J (JaX), C57BL/6NCrl (CRL) [101] Hippocampal mossy fiber morphology C57BL/6JKun (Nijmegen Univ.), C57BL/6JNmg (Nijmegen Univ.) [102] Hippocampal mossy fiber morphology, radial arm maze C57BL/6JKun (Nijmegen Univ.), C57BL/6JNmg (Nijmegen Univ.) [103] Prevalence of molecular-layer heteropia C57BL/6J (JaX), C57BL/6NCrl (CRL), C57BL/6NCrsim (simonsen Lab), C57BL/6Nhla (hilltop Lab.), C57BL/6Nhsd (envigo), C57BL/6NTac (TaC) [104] susceptibility to scopolamine-induced neuronal impairment C57BL/6J (dae han Bio Link), C57BL/6N (dae han Bio Link) [105] Renal/urinary system phenotype degree of glyoxylate-induced kidney crystal deposition C57BL/6J (JaX), C57BL/6NCrl (CRL) [106] Respiratory system phenotype airway responsiveness C57BL/6J (JaX), C57BL/6NCrl (CRL), C57BL/6NTac (TaC), C57BL/6Nhsd (envigo), C57BL/6NCr (Nih) [107] Response to neonatal hyperoxia-induced lung injury C57BL/6J, C57BL/6N [108] skeleton phenotype Bone structure and unloading-induced bone loss in adolescents C57BL/6J (hJaX), C57BL/6NJ (JaX) [109] Trabecular bone mass C57BL/6J (JaX), C57BL/6Jolahsd (envigo), C57BL/6JRcchsd (envigo) [110] Vision/eye phenotype Constriction during pupillary light response with aging C57BL/6J, C57BL/6N [111] ocular dominance plasticity C57BL/6J (JaX), C57BL/6Jolahsd (envigo) [112] susceptibility to laser-induced choroidal neovascularization C57BL/6J (JaX), C57BL/6NTac (TaC) [113] susceptibility to photoreceptor oxidative stress C57BL/6J (JaX), C57BL/6NCrl (CRL) [114] Comprehensive phenotype* eMPRessslim pipelines C57BL/6J (JaX), C57BL/6NTac (TaC) [115] The phenotype terms column basically follows the Mammalian Phenotype ontology [116] . The parentheses to the right of the strain names indicate the vendor or facility where the mice were produced. JaX, The Jackson Laboratory; CRL, Charles River Laboratories; TaC, Taconic Biosciences; Wehi, Walter and eliza hall institute; hZM, helmholtz Zentrum München; CeMiB, Multidisciplinary Center for Biological investigation on Laboratory animal science, The University of Campinas. strains without parentheses are those for which information on the vendor or production facility was not included in the publication. *By comprehensive phenotype screening (substrain differences were identified in cardiovascular, hematopoietic, adipose tissue, behavioral/neurological, renal/ urinary, skeletal, hematopoietic, vision/eye, and immune system phenotypes).

Exp. Anim. 2021; 70 (2): [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] differences in prevalence of sexual dimorphism among C57BL/6 substrains should also be considered. Significant variation in brain injuries has been reported among C57BL/6 substrains [69, 143] . in mouse stroke, male and female C57BL/6N mice show similar infarct sizes, although female C57BL/6J mice tend to have smaller infarcts [100] . Such substrain-specific sexual dimorphisms have also been identified in analyses of behavioral characteristics, serum biochemistry, and immune function [36, 79, 115] . Therefore, in studies of the effects of genetic modifications on sex differences, heterogeneity in substrain backgrounds would likely complicate the interpretation of experimental results. Thus, the phenotypic differences among C57BL/6 substrains are quite numerous, and they should be reported more in the future.

single nucleotide polymorphisms (sNPs) are the most widely used genetic markers in genomes and are useful for uncovering genetic differences between genetically similar lineages, including substrains. in laboratory mouse strains, sNP databases have been developed by mapping sequence data from other inbred lines using the Jackson Laboratory's C57BL/6J strain as a reference [144] [145] [146] [147] [148] [149] . sNPs that distinguish each C57BL/6J substrain can be extracted from candidates specific to the C57BL/6J strain in sNP databases as well as by identify- [125] .

increased rate of sperm morphological abnormalities, unbalanced sex ratio, and dysregulation of several transcripts expressed in the testes

The parentheses to the right of the strain names indicate the vendor or facility where the mice were produced. JaX, The Jackson Laboratory; TaC, Taconic Biosciences. ing the genotypes of candidate sNP loci for each C57BL/6J substrain [6, [150] [151] [152] . in Table 3 The lymphocyte antigen 5 (Ly5) congenic strain is a representative mouse strain derived from the C57BL/6J substrain. The lymphoid cell surface antigen produced by the protein tyrosine phosphatase, receptor type, C (Ptprc; Ly5/Cd45 gene) gene distinguishes between hematopoietic cells from donor and recipient mice; hence, this strain has been widely used as a cell marker for bone marrow transplantation studies [153, 154] . The C57BL/6-Ly5.1 congenic inbred strain (B6JBoy.sJL-Ptptc a ; RBRC00144) was established by Boyes et al. at sloan-Kettering institute through introduction of the Ly5.1 of the sJL/J strain into the C57BL/6JBoy strain background (Ly5.2 allele) by 23 serial backcrosses [155, 156] . The exact point at which the C57BL/6JBoy strain diverged from the C57BL/6J strain is not clear, but the literature suggests that divergence occurred in the 1970s, or possibly even earlier [157] . according to genetic background testing using C57BL/6J-specific SNP markers, the genetic background should be similar to that of older C57BL/6J substrains, which have an earlier divergence date (Table 4 ). although C57BL/6-Ly5 antigen congenic mice are currently available from several vendors, a spontaneous mutation in the natural cytotoxicity triggering receptor 1 (Ncr1) locus and residual chromosomal fragments of the donor SJL/J strain, both of which affect immune response, have been reported [158, 159] . These effects must be considered to properly interpret the results of experiments involving these strains [158, 159] .

C57BL/6J 0 a/a

in the C57BL/6N substrain, genotyping of the above C57BL/6J-specific SNP loci could not detect polymorphism as observed in C57BL/6J [6] . This is because the previous C57BL/6J-specific SNP information was obtained by mapping the abundant reference sequence data of C57BL/6J mice to the sequences of other strains. however, there was a lack of previous sequence data for C57BL/6N, and thus the detection of C57BL/6N strainspecific SNPs was infeasible. Later, in 2011, a re-sequencing analysis of the C57BL/6NJ strain was performed [160, 161] , and highly accurate sequence information was published on the website of the sanger institute (hinxton, United Kingdom; https://www.sanger. ac.uk/sanger/Mouse_snpViewer/rel-1505). The results enabled a subsequent search for C57BL/6N strain sNPs and revealed SNP loci specific to the C57BL/6NJ strain and their genotypes in seventeen C57BL/6 substrains [7] . There were 86 polymorphic sNP loci at regular in-tervals on every chromosome, selected from approximately 1,400 C57BL6/NJ strain-specific SNP candidate loci. as results similar to those of the C57BL/6J group, the number of C57BL/6N-specific SNPs was correlated with the branching year and breeding history of each lineage (see Fig. 1 in Mekada et al. 2015 [7] ). The previous information required for genotyping each sNP locus listed in Mekada et al. 2015 [7] was updated with additional primer information for allele-specific PCR, and it is listed in supplementary Table 3 .

our research group continues to search for sNPs that can distinguish among various C57BL/6 substrains; such information is expected to improve our understanding of the genetic background of these strains. in addition to sNPs, dNa polymorphisms such as indels and short tandem repeats as well as structural variants, including C57BL/6-specific genetic variants found in Nnt and Snca genes, can be used to distinguish C57BL/6 strains in terms of genetic background [14, 115, 162] . These dNa polymorphism markers may be useful for comparing substrains and can also be applied to gene mapping involving the crossing of C57BL/6 substrains. a genetic screening model based on these principles would enable the mapping of polymorphic loci that contribute to the variability of a trait among strains with a largely homogenous background; this could lead to improvements in mapping resolution and aid in the selection of candidate genes. To date, numerous quantitative trait loci have been identified, including genes associated with susceptibility to heat nociception [53] , diet-induced obesity [84] , dominant obesity [163] , circadian activity [164] , feeding disorder [37] , response to cocaine [41] , defective neutrophil recruitment [91] , and non-ectopic endogenous retroviral dysregulation [92] . With advances in genome sequencing technology, genomic information for inbred mice, including substrains, is being continually updated [165, 166] . It is expected that many new strain-specific haplotypes will be identified in the future. 

1989 a/a a/a a/a G/G T/T a/a a/a a/a T/T T/T G/G T/T C/C T/T a/a C/C a/a T/T T/T G/G C57BL/6JMs 1984 a/a C/C a/a G/G T/T a/a a/a a/a T/T T/T G/G T/T C/C T/T a/a C/C a/a T/T T/T G/G C57BL/6JeiJ 1976 a/a C/C a/a G/G T/T a/a a/a a/a T/T T/T G/G T/T C/C T/T G/G C/C a/a T/T C/C G/G C57BL/6Jolahsd 1974 a/a C/C a/a C/C T/T a/a a/a a/a T/T T/T G/G T/T C/C T/T G/G C/C a/a T/T C/C G/G C57BL/6JBomTac 1971 T/T C/C a/a C/C T/T a/a a/a a/a T/T T/T G/G C/C C/C T/T G/G C/C a/a T/T C/C G/G C57BL/6JNrs 1965 T/T C/C a/a C/C C/C a/a a/a G/G T/T T/T G/G C/C C/C T/T G/G T/T G/G T/T C/C C/C B6JBoy.sJL-Ptprc a [Ly5.1] T/T C/C a/a C/C C/C a/a a/a G/G T/T T/T G/G C/C C/C T/T G/G T/T G/G C/C C/C C/C B6JBoy.RF-Cd8 a Cd8b1 a [Ly2. 1, Ly3.1] T/T C/C a/a C/C C/C a/a a/a G/G T/T T/T G/G C/C C/C T/T G/G T/T G/G C/C C/C C/C C57BL/6N substrains 1951 T/T C/C a/a C/C C/C G/G G/G G/G C/C C/C a/a C/C T/T C/C G/G T/T G/G C/C C/C C/C *The year in which each C57BL/6 substrain branched from the C57BL/6J strain of the Jackson Laboratory. † Corresponds to Locus No. in supplementary Table 2 .

C57BL/6J substrains have been the most widely used background for many mutant strains, including genetically-modified lines. another major lineage, the C57BL/6N substrain, has been used as the genetic background for embryonic stem cells in large-scale generation of knockouts [12] . as a result, many mutant mice derived from the C57BL/6J and C57BL/6N substrains are used worldwide, including in Japan. however, given that the substrains are widely considered interchangeable, significant phenotypic differences among substrains are often overlooked. The phenotypic differences among C57BL/6 substrains reported to date should be considered by researchers. For example, certain substrains should be used with caution in conditional knockout models because conditional knockout mice should be crossed with Cre and/or Flp recombinase mice. Combining recombinase strains with different genetic backgrounds might produce knockout animals with mixed genetic backgrounds, leading to unexpected phenotypic variations. When the genetic backgrounds of experimental mice are not identical, erroneous conclusions are likely to be drawn. if a suitable substrain cannot be used, wild-type littermates might serve as an appropriate control group. Recently, it has become clear that phenotypic differences among substrains are caused by a complex combination of genetic and microbial alterations [167, 168] . Use of wild-type littermates might standardize the microbiota, thereby facilitating investigation of the phenotype or function of specific genes that could be affected by the microbial community [169, 170] .

Today, the rise in prominence of genome editing technologies has made it possible to directly generate various types of genetically modified mice, including gene knockout, conditional knockout, and gene knock-in, by using the fertilized eggs of inbred mice without the need for embryonic stem cells. at present, more than 50% of the strains deposited at the RiKeN BRC have been generated by genome editing using fertilized eggs from inbred strains, although not necessarily C57BL/6 mice. Just as there are C57BL/6 substrains, there are also substrains of other inbred lines, and as is the case with the C57BL/6 strain, an accumulation of genetic and phenotypic differences is to be expected. More recently, many mouse models have been generated for coronavirus research; the BaLB/c mouse strain is known to be popular in the immunology field and susceptible to SARS-CoV-2 [171, 172] , and it is increasingly being used as a back-ground strain for CoVid-19 research [173] [174] [175] . The BaLB/c strain has a long history in the form of the above B6 mice [2, 3] . at least 16 BaLB/c substrains, including BaLB/cJ, BaLB/cBy, and BaLB/canN, available to the research community can be found by performing a PubMed research with the search term "BaLB/c". Furthermore, several papers have reported phenotypic variations in infection and immune responses [176] [177] [178] . Therefore, we should pay much more attention to the substrain status of BaLB/c mice in research on CoVid-19, which is increasing around the world.

Conclusion experimental data must be robust, reliable, and reproducible; only under these circumstances can science progress [179] . To ensure experimental reproducibility and minimize bias, researchers should recognize potential phenotypic differences among substrain mouse models, select appropriate substrains for their own research purposes, and share the full names of the substrains used with the scientific community. In the future, it is expected that journals will adopt a policy requiring a detailed description of the substrain and the breeding history of model mice used in research.

We are grateful to Masayo Kadota, Mayu hirose, ayumi Murakami, and the other members of the experimental animal division, RiKeN BRC, for their devoted technical assistance. We also thank ikuo Miura, Kuniya abe, Toshihiko shiroishi, Yuichi obata, and Kazuo Moriwaki of RiKeN BRC for their valuable advice and support. This work was supported by a Grantin-aid for Young scientists (B) from the Ministry of education, Culture, sports, science and Technology (MeXT) of Japan (Grant Number 23700518) to K.M. The RiKeN BioResource Research Center has been a participant in the National BioResource Project (NBRP) organized by MeXT and designated as the core facility for mouse resources of the NBRP.

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