A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Abstract

Background: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

Figure 1

Heat maps illustrating significant differences in phenotype parameters between C57BL/6N and C57BL/6J male and female mice. Parameters were assessed from each of the four centers: Helmholtz Zentrum Munchen (HMGU), Institut Clinique Souris (ICS), MRC Harwell, and the Wellcome Trust Sanger Institute (WTSI). Parameter designations and parameter descriptions are from EMPReSSslim [37]. Significance levels and the direction of the effect (red and green) are defined in the key. Significant differences for categorical data are illustrated in blue. (A, B) Phenotype parameters showing a significant difference between N and J in (A) three or more centers, and (B) in two centers but no evidence of trends in the other centers.

Figure 2

Heat maps illustrating significant differences in phenotype parameters between C57BL/6N and C57BL/6J male and female mice. Parameters were assessed from each of the four centers (Helmholtz Zentrum Munchen (HMGU), Institut Clinique Souris (ICS), MRC Harwell, and the Wellcome Trust Sanger Institute (WTSI)). Phenotype parameters that showed significant differences in two or more centers, but the opposite trend in another center. Parameter designations and parameter descriptions are from EMPReSSslim. Significance levels and the direction of the effect (red and green) are defined in the key.

Figure 3

Morphological and functional differences between C57BL/6N and C57BL/6J mice eyes. (A) Whereas white flecks were absent from C57BL/6J fundus, they were frequently detected in the ventral retina from C57BL/6N mice, with various degrees of severity, as illustrated here by three C57BL/6N fundus images. Depending on the investigating center, C57BL/6N mice with at least one eye affected represented 69.2% (n = 70 males + 34 females; ICS), 44.6% (n = 145 males + 158 females; GMC), or 23.0% (n = 184 males + 194 females; WTSI) of the population, whereas no flecking was detected in C57BL/6J mice (ICS: 29 males; GMC: 75 males + 75 females; WTSI: 34 males + 28 females). (B) Both vein and artery numbers differed from mouse to mouse in both strains, usually being between (left) three and (right) seven, with (middle) a mean of around five, as can be seen in three fundus images from C57BL/6J mice. (C) Quantification of veins and arteries in male C57BL/6N and C57BL/6J mice (n = 140 and n = 70 eyes, respectively). The mean number of veins per eye was 4.8 ± 0.1 for C57BL/6N (n = 122 eyes) versus 5.3 ± 0.1 for C57BL/6J mice (n = 138 eyes). Both differences were significant (P < 0.001, Mann-Whitney rank sum test).

Figure 4

Micro-computed tomography (μCT) analysis of distal femur showed similar trabecular bone parameters in 14-week-old C57BL/6J and C57BL/6N mice. (A) Males and (B) females. (C) Cortical bone parameters from midshaft femur of 14-week-old male mice were also unchanged between the two strains. (D) Measurement of serum osteocalcin and urinary deoxypyridinoline (bone formation and bone resorption markers, respectively), indicates that bone turnover was identical between 14-week-old C57BL/6J and C57BL/6N. Abbreviations: BV/TV, bone volume/tissue volume; TbN, trabecular number; TbSp, Trabecular spacing; Conn-Dens, Connectivity density; SMI, structural model index (0 for parallel plates, 3 for cylindrical rods); DA, degree of anisotropy; CtPo, cortical porosity; CtTh, cortical thickness; DPD, deoxypyridinoline; creat, creatinin.

Figure 5

Light/dark test. Bars represent (A) the latency to enter, (B) the percentage of time spent in the dark compartment (C) and the number of light/dark transitions by C57BL/6J (n = 10) and C57BL/6N (n = 9) male mice, aged 8 to 10 weeks. Data are mean ± SEM, *P < 0.05 (t-test). (D) Rotarod motor learning performance over 4 days. Symbols and lines represent daily latencies (mean ± SEM) to fall from rotating rod at acceleration from 4 to 40 rpm in 300 seconds by C57BL/6J (n = 10) and C57BL/6N (n = 10) male mice, aged 9 to 11 weeks. *P < 0.05, **P < 0.005, ***P < 0.0001 J versus. N (t-test); °P < 0.05, °°P < 0.005, °°°P < 0.0001 versus day 1, Fisher's (least squares difference).

Figure 6

Morris water maze. (A) Learning curves for familiarization and training phases Symbols and lines represent daily latencies (mean ± SEM) to reach the platform by C57BL/6J (n = 10) and C57BL/6N (n = 10) male mice, aged 16 to 20 weeks. **P < 0.005, ***P < 0.0005 J versus N (t-test); °°P < 0.005, °°°P < 0.0001 versus day 1 (Fisher's least squares difference). (B) Probe test. Bars represent % time spent in each quadrant on day 5 during probe test. Dotted line is set at chance level (25%). *P < 0.05, **P < 0.005, ***P < 0.0005 versus correct quadrant (t-test)_. (C) Representative tracks of two C57BL/6J and C57BL/6N mice paths during probe test. Dotted circle indicates former platform location.

Figure 7

Comparison of Listeria host resistance between C57BL/6J and C57BL/6N inbred strains. (A) Kaplan-Meier survival curves of females and males of the C57BL/6J and C57BL/6N strains after intravenous (IV)v. infection with 2 × 10⁴ colony-forming units (cfu) of Listeria monocytogenes strain EGD. (B) Bacterial load in liver and spleen of C57BL/6J and C57BL/6N mice after IV infection with 2 × 10⁴ cfu L. monocytogenes EGD. Organ loads were ascertained at four time points to analyze kinetics of bacterial growth. (C) Comparison of plasma levels of interleukin (IL)-6, interferon-inducible protein (IP)-10, and chemokine ligand (CCL)2 between the C57BL/6J and C57BL/6N mice shown in (B). Concentrations of pro-inflammatory cytokines and chemokines were determined in peripheral blood samples using the Cytokine Mouse 20-Plex Panel (Invitrogen Inc., Foster City, CA, USA) and a LiquiChip 100 system (Qiagen, Hilden, Germany). Significant differences are indicated as follows: *P < 0.05, **P < 0.01 Mann-Whitney, U-test. Black bars and symbols, C57BL/6J inbred strain. White bars and symbols, C57BL/6N inbred strain.

Figure 8

Measurement of splenic natural killer (NK) cells and hapten-specific hypersensitivity. (A) Splenic NK cell activity of C57BL/6J (B6J) versus C57BL/6N (B6NTac) mice: (upper panel) male and (lower panel) female. Splenic NK cells from C57BL/6J or C57BL/6N mice were stimulated under the indicated conditions (six mice per group). Mean ± SD of interferon (IFN)γ-positive cells among a population of CD3- NK1.1+ NK cells was measured by flow cytometry. (B) Hapten-specific hypersensitivity. Male or female C57BL/6J or C57BL/6N mice were sensitized by the application of 25 μl of 0.5% dinitrofluorobenzene (DNFB) solution on the ventral skin. They were then challenged by the application of 5 μl of 0.15% DNFB solution on the left ear 5 days later (DNFB group). The right ears were painted with vehicle (-) and used as controls. Ear thickness was measured 48 hours after challenge. Results are representative of three independent experiments with six mice per group. *P < 0.05; **P < 0.005 (Mann-Whitney U-test).