C57BL/6 Substrain Differences
Understanding the differences between C57BL/6 substrains is essential for experimental design. The two major substrains, C57BL/6J and C57BL/6N, diverged decades ago and carry distinct genetic variants that affect metabolism, behavior, and other phenotypes.
C57BL/6J (Jackson Substrain)
C57BL/6J is the original Jackson Laboratory substrain and the reference for the mouse genome sequence. Key characteristics include:
- Nnt mutation: Spontaneous deletion in nicotinamide nucleotide transhydrogenase affects mitochondrial redox balance and insulin secretion
- Metabolic phenotype: Impaired glucose stimulated insulin secretion; more susceptible to diet induced glucose intolerance
- Behavioral phenotype: Lower anxiety in some paradigms compared to C57BL/6N
- Historical prevalence: Most widely used substrain historically; extensive phenotypic literature
The Nnt mutation in C57BL/6J should be considered when studying metabolic phenotypes, particularly those involving pancreatic beta cell function or oxidative stress.
C57BL/6N (NIH Substrain)
C57BL/6N originated from the NIH colony and is the substrain used by the International Knockout Mouse Consortium (IKMC). Key characteristics include:
- Intact Nnt: Functional nicotinamide nucleotide transhydrogenase; normal mitochondrial function
- Metabolic phenotype: More robust glucose stimulated insulin secretion compared to C57BL/6J
- Crb1 mutation: Retinal degeneration allele (rd8) present in some C57BL/6N colonies; causes retinal lesions
- IKMC compatibility: ES cells and knockout alleles from EUCOMM/KOMP are on C57BL/6N background
C57BL/6N is often preferred for metabolic studies due to intact Nnt, but researchers should verify rd8 status when studying retinal or visual phenotypes.
Choosing Between Substrains
Consider the Nnt status when studying metabolic phenotypes. Consider rd8 when studying retinal or visual phenotypes. For projects using IKMC alleles, C57BL/6N maintains pure background without backcrossing.
Why C57BL/6 for Gene Targeting
Research Community Standard
C57BL/6 is the most commonly used background for genetically engineered mouse models. This widespread adoption provides:
- Extensive baseline phenotypic data for comparison
- Compatibility with published Cre driver lines
- Straightforward literature comparison across laboratories
- Well characterized responses to common experimental paradigms
Cre Driver Compatibility
The majority of tissue specific Cre driver lines are maintained on C57BL/6 backgrounds. Generating conditional alleles on C57BL/6 enables direct crosses to Cre drivers without introducing mixed background effects.
Tissue Specific Cre LinesApplications by Research Area
Metabolic Research
Strain background significantly impacts metabolic phenotypes:
- Diet induced obesity: C57BL/6J develops more pronounced obesity and glucose intolerance on high fat diet
- Insulin secretion studies: C57BL/6N preferred when studying beta cell function due to intact Nnt
- Diabetes models: Background choice affects baseline glucose homeostasis and disease susceptibility
Document substrain in publications and consider Nnt genotype when interpreting metabolic data.
Neuroscience Research
Behavioral and neurological phenotypes vary between substrains:
- Anxiety related behavior: Substrains differ in open field and elevated plus maze responses
- Learning and memory: Subtle differences in some cognitive paradigms
- Retinal studies: Verify rd8 status in C57BL/6N when studying visual system
Immunology Research
C57BL/6 mice carry the H2b MHC haplotype and display Th1 biased immune responses:
- Well characterized immune cell populations
- Extensive reagent availability (antibodies, tetramers)
- Compatible with most syngeneic tumor models
Oncology Research
C57BL/6 is compatible with common syngeneic tumor cell lines and provides immunocompetent background for immuno oncology studies:
- B16 melanoma, MC38 colon carcinoma, LLC lung carcinoma compatibility
- Suitable for immune checkpoint studies
- Well characterized tumor microenvironment responses
Technical Considerations
Backcrossing Requirements
When targeted alleles are generated on 129 strain backgrounds, backcrossing to C57BL/6 is required to achieve congenic status. Speed congenic approaches using marker assisted selection can achieve N10 equivalent purity in fewer generations.
Backcrossing ServicesBreeding Performance
C57BL/6 mice have moderate breeding performance compared to outbred strains:
- Average litter size: 5 to 7 pups
- Weaning age: 21 days
- First litter typically at 10 to 12 weeks of age
- Good maternal behavior
Plan breeding timelines accounting for these parameters when estimating cohort development schedules.
Health and Husbandry
C57BL/6 mice are generally robust with good health profiles:
- Compatible with SPF housing conditions
- Moderate lifespan (approximately 24 to 30 months)
- Low incidence of spontaneous tumors at typical experimental ages
- Susceptible to age related hearing loss (Ahl locus)
Backcrossing Generations
Selected Publications
Models on C57BL/6 background generated by ingenious targeting laboratory:
Salzbank J, Lacaille H, Gaby J, O'Reilly JJ, Kissner M, Vacher CM, Penn AA. 2025. Microglia alter sex-specific cerebellar myelination following placental hormone loss. Nat Commun. 16(1): 9846
Zhou W, Zhang J, Chowdhury NU, Norlander AE, Toki S, Abney M, Rusznak M, Gibson-Corley KN, Cook DP, Newcomb DC, Peebles RS Jr. 2025. PGI2 signaling metabolically reprograms CD4 Th2 cells and represses allergic airway inflammation. J Immunol 9(214): 2270-2280
What Researchers Say
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University of Pennsylvania
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Yale University
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Warren Center for Neuroscience Drug Discovery
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— Albert Basson, PhD
King's College London
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