The Past of C57BL/6 Mice

This dark gray coat rodent, with its unquiet gene background and biology characteristic, have become the "frequenter" of global lab. Whether immunity, exploration of Metabolic Diseases, or neuroscience, the presence of C57BL/6 mice is everywhere. I'll uncover the mysterious veil of C57BL/6 mice by this article.

C57BL/6 Background

In 1921, the researcher C.C.Little bred several inbred lines via Abby Lathrop mice, the male mice number 57 matched with the female mice number 52 to cultivate C57. The dark gray coat mice was cultivated into C57BL. BL is the abbreviation of "Black". In 1937, Little separated the C57BL paternal line into C57BL/6 (Group 6 Sublineage) and C57BL/10 (Group 10 Sublineage).

The C57BL/6J and C57BL/6N mice commonly used in scientific research were bred in 1937 and 1951 respectively. With the investment of different research institutions, the strains of C57BL/6 mice have expanded to more than 20 types. Figure 1 lists the origins of some strains.

Fig.1 History of C57BL/6 substrain.¹

C57BL/6N vs C57BL/6J

C57BL/6 has numerous distinct sublineages. Among them, C57BL/6N and C57BL/6J stand out, and it's crucial to recognize the disparities between these two. Why is this important? Well, when we're in the process of selecting an experimental animal, these differences can significantly impact the outcomes of our research. Since C57BL/6N and C57BL/6J are frequently-employed sublineages, it begs the question: precisely what sets them apart from each other?

Gene sequence differences

The genetic sequence differences between C57BL/6J and C57BL/6N mice are mainly concentrated in the coding region SNPs, small insertions/deletions (indels), and structural variations (SVs). The specific information is as follows:

(1) 34 coding SNPS: there have 32 missense mutations, 1 nonsense mutation (Spata31 gene, leading to premature termination), and 1 splicing mutation (Armcx4 gene).

(2) There are still have 2 coding indels, both are frameshift mutations. Such as the Crb1 gene (1 bp deletion, specific to the N strain, associated with retinopathy).

(3) Structural Variation (SVs): These structural variations are mainly 15 SVs overlapping genes, including 2 coding region variations (Vmn2r65 deletion, Nnt exon deletion) and 13 non-coding region variations (such as intron LINE insertion). The Cyp2a22 gene has a complete gene insertion (LINE element) in the J strain, which may affect metabolic function.

Here is a simple summary of some specific mutations and corresponding functions of the two strains of mice.

The differences of eyes

Comparing with C57BL/6J, C57BL/6N has decreased vision. We can find the white spots in the fundus of C57BL/6N, but not in C57BL/6J. The average number of arteries and veins in C57BL/6J is greater than that in C57BL/6N. In addition, C57BL/6J mice are prone to microphthalmia and other related eye diseases. You can discover the differences between the two sublineages from the following pictures (fig. 2).

Fig.2 Morphological and functional differences between C57BL/6N and C57BL/6J mice eyes. (A) the fundus of C57BL/6J. (B) the fundus of C57BL/6N.

Metabolic and endocrine differences

Due to the deletion of the Nnt gene (exon 7-11) in strain J, glucose tolerance was significantly impaired (abnormal IPGTT curve), but the basal blood glucose of mice in this strain was normal. However, the Pmch missense mutation (isoleucine → threonine) of the N strain has a higher oxygen consumption and slightly higher basal blood glucose, but the glucose tolerance of the N strain is normal. There are also obvious differences in energy metabolism between these two strains. The resting metabolic rate (calorie production) of J strain mice is lower (especially in females), and their nocturnal activity and food intake are reduced. The corresponding N strain has higher fat quality and lower lean meat quality (DEXA test).

Neurobehavioral differences

Firstly, in terms of movement and learning, the N strain has a short latency (poor motor coordination) in the Rotarod test and a long latency in the Morris water maze test. This might be due to impaired spatial memory or the influence of vision. This means that the J strain has a greater advantage in behavioral experiments. For instance, in the field experiments, the J strain mice showed higher motor activity (more entries in the open-field experimental center), Rotarod performed outstandingly, and their learning ability improved rapidly.

There are also differences in emotions and feelings. Mice of the N strain had a longer latency period when entering the dark room (anxied-like behavior) in the light-dark experiment, a lower amplitude of sonic shock reflex, but a higher pre-pulse inhibition (PPI). The mice of strain J showed no difference in anxiety, had a stronger sonic shock reflex and normal PPI.

Characteristics and Application Differences of C57BL/6N and C57BL/6J Mouse Strains

In conclusion, we should select different strain based on specific studies. For instance, mice of strain J are more suitable for studies on glucose metabolism (such as diabetes models), exercise capacity and immune inflammation, but attention should be paid to the interference of Nnt deficiency. Mice of the N strain are more suitable for studies on retinopathy, neurobehavior (such as anxiety), and infection immunity. The influence of Crb1 rd8 on vision-related experiments needs to be excluded. Next, I will respectively sort out the research that is more suitable for the two strains of mice.

C57BL/6N Mouse Strain

C57BL/6N is one of the most widely used inbred mouse strains, frequently serving as the genetic background for transgenic or knockout models in genetic engineering. Its key biological features and research applications are as follows:

(1) Genetic and Behavioral Traits

Behavioral Distinctions: When compared to the C57BL/6J strain, the C57BL/6N strain shows notable differences in behavioral phenotypes. It exhibits more frequent grooming behaviors, like barbering. This characteristic makes it a suitable animal model for studying compulsive hair-pulling disorders.

Intact Nnt Gene: The C57BL/6N strain retains the wild-type nicotinamide nucleotide transhydrogenase (Nnt) gene. Unlike some other strains, it doesn't have exon deletions. This intact gene helps maintain normal regulation of glucose metabolism homeostasis.

(2) Physiological and Pathological Features

Auditory System: Due to the Ahl gene deletion, C57BL/6N shows late-onset hearing loss, with age-related increases in auditory thresholds and structural of inner and outer hair cells, reducing susceptibility to audiogenic seizures.

Metabolic Sensitivity: Valuable for studies on obesity, metabolic syndrome, and type II diabetes, its stable phenotype provides a reliable model for mechanistic research.

(3) Disease Model Applications

Addiction and Alcohol-related Diseases: The C57BL/6N strain has a relatively low aversion to alcohol. After consuming alcohol, it readily shows higher plasma alcohol concentrations. This makes it an apt model for studying alcoholic diseases such as fatty liver, pancreatitis, and osteonecrosis of the femoral head. It's also useful for research on morphine addiction.

C57BL/6J Mouse Strain

Derived as an inbred subline from C57BL, C57BL/6J is an ideal model for metabolic diseases, aging, and cardiovascular research due to its unique genetic variations:

(1) Genetic and Metabolic Traits

Nnt Gene Mutation: The C57BL/6J strain has a rather unique characteristic-a natural deletion of exons 7-11 in the Nnt gene. This deletion doesn't happen without consequences. It disrupts the normal function of nicotinamide nucleotide transhydrogenase. As a result, insulin secretion takes a hit, leading to mild-to-moderate hyperglycemia. This set of events makes the C57BL/6J strain a classic model for type II diabetes research. It's like nature provided researchers with a ready-made tool to study this complex metabolic disorder.

Diet-Induced Obesity (DIO) Sensitivity: The C57BL/6J strain is highly responsive to high-fat diets. When you put these mice on a high-fat diet, it's almost like flipping a switch. They quickly develop obesity, insulin resistance, and hyperinsulinemia. This makes them a primary choice for modeling metabolic syndrome and fatty liver. Researchers can observe and study how the body responds to excessive fat intake in a relatively short time frame.

(2) Physiological and Pathological Features

Auditory and Skeletal Systems: Similar to C57BL/6N, it experiences age-related hearing loss due to Ahl gene defects, though noise exposure accelerates this process; it also has lower bone density, requiring consideration in osteoporosis-related research.

Developmental Phenotypes: Higher incidence of microphthalmia, hereditary hydrocephalus (1–4%), and malocclusion compared to other sublines, necessitating close attention in related phenotypic studies.

(3) Disease Models and Longevity

Cardiovascular Diseases: The C57BL/6J strain carries eight genes (Ath1 to Ath8) that are related to atherosclerosis. These genes predispose it to atherosclerotic plaque formation. In the world of cardiovascular research, this makes it a core model. Scientists can use it to study how these plaques form and develop, which is crucial for understanding and potentially treating cardiovascular diseases.

Aging and Oncology: When it comes to aging and oncology research, the C57BL/6J strain has some interesting features. The primitive hematopoietic stem cells in these mice senesce more slowly. Also, they have a lower spontaneous tumor incidence compared to other strains, and they tend to live longer. All these factors combined make them a great option for studying the aging mechanism and anti-tumor research. It's like they offer a window into the processes that govern aging and tumor development.

Addiction Studies: Like C57BL/6N, its low alcohol aversion supports modeling alcoholic diseases (e.g., liver fibrosis, intestinal mucosal hyperpermeability) and morphine addiction.

Strain Selection Recommendations

When we selecting C57BL/6 substrains for experimental design, consider the following key differences aligned with research goals:

Metabolic and Endocrine Research: For studies focused on diabetes, obesity, and metabolic syndrome, C57BL/6J is a great choice. The reason lies in its Nnt mutation. This genetic alteration makes it an ideal model for mimicking these metabolic conditions. On the other hand, if you're interested in studying glucose homeostasis mechanisms or need a control group, C57BL/6N is a suitable option. It has the wild-type Nnt gene, which allows for a more "normal" baseline in glucose-related research.

Neurobehavioral and Auditory Studies: When it comes to neurobehavioral and auditory research, you'll want to screen the substrains based on specific phenotypes. For example, if you're looking into compulsive behaviors, C57BL/6N is notable for its more frequent compulsive grooming. In contrast, if age-related hearing loss is your area of focus, C57BL/6J fits the bill as it exhibits this characteristic.

Environmental and Genetic Control: Both strains exhibit age-related hearing loss, but C57BL/6J is more sensitive to noise, requiring strict environmental control in auditory function experiments.

Overall, researchers should integrate target phenotype genetics (e.g., Nnt, Ahl status), strain-specific pathological features, and literature data to select the appropriate mouse strain, ensuring research reliability and reproducibility.

References

1. Song, Hyun Keun, and Dae Youn Hwang. "Use of C57BL/6N mice on the variety of immunological researches." Laboratory animal research 33 (2017): 119-123. https://doi.org/10.5625/lar.2017.33.2.119
2. Simon, Michelle M., et al. "A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains." Genome biology 14 (2013): 1-22. https://doi.org/10.1186/gb-2013-14-7-r82
3. Sarsani, Vishal Kumar, et al. "The genome of C57BL/6J "Eve", the mother of the laboratory mouse genome reference strain." G3: Genes, Genomes, Genetics 9.6 (2019): 1795-1805. https://doi.org/10.1534/g3.119.400071
4. Distributed under Open Access license CC BY 4.0, without modification.

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