Fernando Benavides DVM, PhD, Laboratory Animal Genetic Services, Department of Epigenetics and Molecular Carcinogenesis, The University of Texas - M.D. Anderson Cancer Center
When I ask postdocs or students which particular substrain of a common inbred strain they are using, I often get that “what are you talking about?” face. I want to create an awareness of the existence of a variety of substrains of mice that, if ignored, could affect the results and/or reproducibility of some experiments. Most mouse users are not fully aware that inbred strains of mice and rats are actually distributed in “families” of related substrains. These substrains stem from a common ancestral strain and present several genetic differences.
While permanent inbreeding effectively eliminates a proportion of new mutant alleles, another undetected fraction may become progressively fixed in the homozygous state, replacing the original allele. This occurrence is known as genetic drift. Genetic drift contributes inexorably to strain divergence and the generation of substrains when the same strain is propagated independently in different places 1-2.
Mutations hidden in the genomes of substrains are sometimes referred to as passenger mutations 3. Most of the passenger mutations carried by substrains are quiet 4. As a result, there is no obvious phenotype stemming from the mutation. Some genetic differences are just variant alleles (polymorphisms) for genetic markers like microsatellites (SSLPs) and SNPs (single nucleotide polymorphism). However, there are many examples in the literature where substrains originating from the same inbred strain have acquired new phenotypes as a consequence of genetic drift 5-7.
Naming and describing inbred strains and substrains with standard nomenclature is critical8-9. The International Committee on Standardized Genetic Nomenclature for Mice and Rats established rules guiding nomenclature that are continuously updated. These rules, last revised in January 2016, are described on the MGI webpage under 'Guidelines for Nomenclature of Mouse and Rat Strains' (http://www.informatics.jax.org/mgihome/nomen/strains.shtml). A helpful and visual Mouse Nomenclature Quick Guide is available at https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genetics-and-nomenclature#
Substrains: All in the Family?
A particular colony of an inbred strain can be considered a substrain in the following cases: (i) when residual heterozygosity (incomplete inbreeding) is still present at the time of separation; (ii) when undetected spontaneous mutations become fixed in a colony (genetic drift); and (iii) when a strain is separated from its parent colony for a total of 20 or more consecutive generations of inbreeding (e.g., parent colonies have each been bred for 10 generations). The latter is the more common situation, and it is not necessary to identify genetic differences; simply being separated for more than 20 generations grants the title of substrain (i.e., the use of a particular ILAR approved lab code to identify this new colony) 9.
The C57BL/6 Family
C57BL/6 was historically a common inbred strain. However, since the arrival of the gene targeting technologies, its use has exploded, because it is the most common choice as background for knock-out (KO) and knock-in (KI) lines. C57BL/6J was chosen by the International Mouse Sequencing Consortium as the inbred strain to generate the first draft of the mouse genome (the second mammal to ever be sequenced), published in 2002 10. Interestingly, the number 57 in the name of the strain originated from a female with ID 57 that was crossed by Clarence Cook Little in 1921 with a male with ID 52. The ancestors of C57BL mice were homozygous for the nonagouti allele and therefore exhibited a black coat 11. The same crosses between female 57 and male 52 gave origin to the related strains C57L (leaden coat) and C57BR (brown coat).
In 1929, CC Little was named the first director of the Jackson Laboratory (JAX) in Bar Harbor, Maine, which is still considered the Mecca of mouse genetics. The ancestral group of C57BL/6 (official symbol: B6) mice is the origin of the substrain known today as C57BL/6J (J is the Lab Code for JAX) (stock #000664). In the last decade, JAX has incorporated a unique (patented), Genetic Stability Program designed to effectively limit cumulative genetic drift in their C57BL/6J substrain by rebuilding their foundation nucleus from pedigreed, cryopreserved embryos every five generations 9 In 2005, they began selling C57BL/6J mice derived from two chosen mice (Adam and Eve mice) through hundreds of frozen embryos of the duo's grandchildren, enough to last for 25-30 years. The complete genome of the above mentioned “Eve” female mouse was recently published12 and showed signs of incipient genetic drift.
Over the years, several substrains have been created due to the distribution of JAX C57BL/6 mice to different places. For example, C57BL/6N (B6/N) was separated from C57BL/6J (B6/J) at generation F32 in 1951, and is the substrain maintained at the National Institute of Health (NIH). ES cells developed from this C57BL/6N substrain are used by the International Knockout Mouse Consortium (IKMC) to create a KO line for every protein-coding gene (~22,000 genes). A comprehensive comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N was recently published 13. During the 1970s, new colonies of C57BL/6N were started in Charles River Laboratories (Charles River) and Harlan (now Envigo). These are the C57BL/6NCrl and C57BL/6NHsd substrains, respectively. The same B6/N substrain was later acquired by Taconic (C57BL/6NTac), Janvier (C57BL/6NRj), and even JAX (yes, JAX also sells the “N” substrain, as indicated by the “NJ” at the end of the name: C57BL/6NJ stock # 005304). In Europe, Envigo distributes the C57BL/KaLwRijHsd substrain, a branch of the original B6/J but separated in 1947 (almost exclusively used as a model for multiple myeloma). In the 1970s and 1980s, other colonies stemming from C57BL/6J were created, like the Eicher substrain (C57BL/6JEiJ), separated in 1976. Other examples are the C57BL/6JolaHsd substrain created in the UK (Harlan Olac) and the C57BL/6JRccHsd substrain in Switzerland (RCC), both distributed exclusively in Europe by Envigo. The C57BL/6JBomTac substrain was started in Germany (Hannover) in the early 1970s and is now distributed by Taconic in Europe. Finally, Charles River also sells C57BL/6J, but only outside the US. As you can see, this is way more complicated than most B6 users around the world suspect. Table 1 lists the mutations present in the C57BL/6 substrains described above, potentially affecting several types of studies, including neurological and behavioral.
The 129 Clan
The 129 group of inbred mice is actually a rather heterogeneous collection of strains that shared a common origin starting in the 1920s. They present genetic variability, including different coat colors and distinctive behavior 14 and in some cases, traces of genetic contamination (accidental crosses). With some differences, 129 mice were recognized for having high incidence of spontaneous testicular teratomas and these strains were the first to be used for the development of pluripotent ES cell lines). Today we distinguish four groups of 129 substrains: named with the letters P (derived from the original parental strains), S (derived from a line carrying the “Steel” mutation), T (derived from a line carrying the teratoma “ter” mutation), and X (derived from a line with a genetic contamination) 15. The nomenclature of the 129 strains were revised in 1997 (http://www.informatics.jax.org/mgihome/nomen/strain_129.shtml).
Many 129 substrains carry the AW (white-bellied agouti) allele and are agouti color with white belly, while others carry mutations on the tyrosinase (Tyr) gene so the agouti color is not expressed due to the absence of melanin. These latter animals show albino or chinchilla coat color (Table 2). Some 129 substrains are homozygous for the Oca2p (pink-eyed dilution) mutant allele which is a model for oculo-cutaneous albinism type 2. To summarize, strains 129X1/SvJ, 129P1, 129P2 and 129P3, are albino or white-bellied, pink-eyed, light chinchilla, but those from the Steel group (129S1, 129S2, 129S3, 129S4, 129S5, 129S6, 129S7 and 129S8) are white- (or light)-bellied agouti (Table 2). Some of the passenger mutations present in the 129 family are: (i) a Cdt1 deletion in some substrains from the P and S group 16 (ii) a missense mutation in the Slc3a1 gene in 129S2/SvPas 17 (iii) a Disc1 deletion in 129S6/SvEv 18 and (iv) a deletion in the Casp4gene in 129X1, 129S1, 129S2, 129S6 and 129P3 3.
The BALB/c Tribe
The BALB (Bagg albino) strain was started by Halsey J. Bagg in New York using albino mice from a pet dealer in Ohio around 1913 and later inbred by McDowell. In the 1930s, the future Nobel laureate George Snell took BALB/c mice to JAX (and added the lower-case c, the symbol for the albino mutation), and this provided the basis for the current BALB/cJ mice and several BALB/c substrains. The lineage giving rise to the Andervont (An) and Bailey (By) substrains and the one originating BALB/cJ were separated from the BALB/c maintained by Snell at JAX in the mid 1930s (~F36).
The BALB/cJ substrain has some phenotypic differences with the other BALB/c substrains, including persistent postnatal expression of genes that are normally expressed only in the fetal liver, resistance to atherosclerosis when placed on a high-fat diet, and reduced expression of major urinary proteins. All these features related with a mutation in the Zhx2 gene (Clinkenbeard et al., 2019). Also, BALB/cJ mice are more aggressive than BALB/cAnN and BALB/cByJ.
BALB/cAnN originated from a group of BALB/cJ mice donated to H. B Andervont in 1935 and later transferred to the NIH in 1951. This substrain, with more than 80 years of divergence from the original BALB/c lineage is available from Charles River, Envigo and Taconic (in addition to the BALB/cJBomTac substrain).
The BALB/cBy substrain originates from BALB/cAnN from which it was separated in 1961 and maintained originally by Dr. Bailey. This substrain is homozygous for the Cdh23ahl mutant allele associated with age-related hearing loss (mutation also present in several other inbred strains). BALB/cByJ mice also carry mutations affecting the Acads (Acadsdel-J) and Ahr (Ahrb-2) genes. Similar to BALB/cAnN, BALB/cByJ mice are a good choice to produce monoclonal antibodies (inducing plasmacytomas upon injection of mineral oil). Using the high-density (78,000 SNPs) Mouse Universal Genotyping Array (MegaMUGA), 134 SNPs are different between BALB/cJ and BALB/cBy substrains.
The C3H strain was developed by Leonell C. Strong in the 1920s, crossing albino mice from Halsey Bagg with DBA mice from Little. Several substrains were produced over the years, but the two most common are C3H/HeJ (JAX) and C3H/HeN (NIH). The C3H/HeN branched out from the C3H/HeJ substrain in 1951. The C3H/HeJ substrain carries a spontaneous missense mutation in the Tlr4 gene (toll-like receptor 4 gene, Tlr4Lps-d) making these mice highly susceptible to infection by Gram-negative bacteria 19 On the other hand, C3H/HeN mice carry a wild type allele and responds well to bacterial lipopolysaccharides (LPS). Most of the vendors offer this NIH substrain. When compared with the MegaMUGA SNP array, these substrains show 827 variant alleles.
The original colony started by CC Little in 1909, selecting mice that were homozygous for dilute (Myo5ad), brown(Tyrp1b) and nonagouti (a) alleles (hence the DBA name) was split in 1930, generating the DBA/1 and DBA/2 substrains. These substrains have major differences, probably due to the fact that the original colony presented substantial residual heterozygosity at the time of separation. These substrains even differ at their MHC H2 haplotype (DBA/1 is H2q and DBA/2 is H2d). When tested with the MegaMUGA SNP array, these substrains present 214 differential SNPs. Interestingly, a mutation was identified in the Taar1 gene that only exists in DBA/2J from The Jackson Laboratory, but from Charles River (DBA/2NCrl), Envigo (DBA/2NHsd) or Taconic (DBA/2NTac) 20.
The CBA/J and CBA/CaJ substrains differ in a particular eye phenotype. CBA/J carries the rd1 (Pde6brd1) mutation, hence these mice are blind a few weeks after weaning. On the other hand, CBA/CaJ mice carries a wild-type allele of the Pde6b gene and their vision is normal. In fact, the rd1 mutation is also present in other inbred strains, for example C3H, FVB/N, SJL/J, and SWR/J.
The solution to this potential problem is education. I encourage you to educate yourself on the importance of using the appropriate substrain. Specifically, the use of the same substrain (and vendor) throughout a study. Principal investigators, postdocs, students, and technicians should be educated on these essential aspects of genetic drift and substrains. Finally, standard nomenclature should be carefully followed at every step, from cage cards to publication.
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2 Guenet, J. L., Benavides, F., Panthier, J., Montagutelli, X. Genetics of the Mouse. (Springer, 2015).
3 Kenneth, N. S. et al. An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1. Biochem J 443, 355-359, doi:10.1042/BJ20120249 (2012).
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20 Reed, C. et al. A Spontaneous Mutation in Taar1 Impacts Methamphetamine-Related Traits Exclusively in DBA/2 Mice from a Single Vendor. Front Pharmacol 8, 993, doi:10.3389/fphar.2017.00993 (2017).