Substrains matter in phenotyping of C57BL/6 mice

Abstract

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.

Keywords: C57BL/6; genetic difference; phenotypic difference; single nucleotide polymorphism (SNP); substrain.

Fig. 1.

Genealogy of the C57BL/6 substrains. Dates and relationships are derived from the vendors’ websites and Mekada et al. (2009 and 2015) [6, 7]. Abbreviations: J, The Jackson Laboratory (JAX); Jcl, CLEA Japan, Inc.; Jms, Institute of Medical Science, Japan; Slc, Japan SLC, Inc.; Ms, National Institute of Genetics; Ei, Dr. Eva M. Eicher; Lac, Laboratory Animal Centre (MRC Carshalton Lab Animal Centre); Ola, Olac, Ltd.; Hsd, Harlan Sprague-Dawley, Inc.; CSAL, Centre de Sélection des Animaux de Laboratoire; Rj, Centre D’Elevage R. Janvier; Rcc, RCC Ltd.; Han, Zentralinstitut fur Versuchstierzucht, Hannover; M&B A/S, Taconic’s Bomholtgard, Denmark, facility; Tac, Taconic Biosciences, Inc.; Nrs, National Institute for Quantum and of Radiological Science and Technology; N, National Institutes of Health; Hla, Hilltop Lab Animals, Inc.; Jic, Central Institute for Experimental Animals, Japan; Cr, NCI, DCTD Animal Production Program; Sim, Simonsen Laboratories, Inc.; Crl, Charles River Laboratories; Crlj, Charles River Laboratories Japan; Seac, Seac Yoshitomi, Ltd. (Kyodo Co., Ltd.); By, Dr. Donald W. Bailey. *Derived from embryos frozen in 1984.

Fig. 2.

Summary of a survey on the use of C57BL/6 substrains in Japan. According to information provided by the developer scientists of the 2,939 mouse strains deposited at RIKEN BRC, 49% were crossed with C57BL/6J substrains, including C57BL/6JJcl, C57BL/6JJmsSlc, and C57BL/6J, and 30% were crossed with C57BL/6N substrains, including C57BL/6NJcl, C57BL/6NCrSlc, C57BL/6NCrlCrlj (including C57BL/6NCrl in 35 strains), and C57BL/6NTac. Another 13% were crossed with C57BL/6 substrains of mixed background, and 8% were crossed with uncertain C57BL/6 substrains.

Fig. 3.

The deletion of exons 7–11 in the Nnt gene of C57BL/6J substrains. Genomic polymerase chain reaction (PCR) analysis of exons 6–12 in the Nnt gene showed the absence of PCR products from exons 7–11 in the C57BL/6J, C57BL/6JJcl, C57BL/6JJmsSlc, and C57BL/6JMs substrains. The deletion of the Nnt gene occurred in the C57BL/6J colony of the Jackson Laboratory between 1976 and 1984. The branching years of each strain from the original C57BL/6J strain are given in parentheses. PCR and gel electrophoresis procedures were performed according to the protocol described in Mekada et al. (2009) [6]. PCR product size data are from Huang et al. (2006) [117].