A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains methods
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Comprehensive phenotypic assessment of C57BL/6N and C57BL/6J mice
In parallel to the genomic analyses, the European Mouse Disease Clinic (EUMODIC) consortium has carried out a comprehensive phenotypic comparison of the C57BL/6NTac and C57BL/6J strains. EUMODIC comprises four mouse centers [ ] carrying out broad-based primary phenotyping of 500 mouse mutant knockout lines generated from the European Conditional Mouse Mutagenesis (EUCOMM) and Knockout Mouse (KOMP) projects within the IKMC program. Cohorts of mice from each mutant line enter the E uropean M ouse P henotyping Re source of S tandardised S creens slim (EMPReSSslim) phenotype assessment, which consists of two phenotyping pipelines, together comprising 20 phenotyping platforms (identified by an ESLIM__procedure_number) that are carried out from 9 to 15 weeks (see Additional file, Figure S1). The methods for performing each screen are detailed in the standard operating procedures (SOPs) tha...
Neurological, behavioral, and sensory
We conducted a number of tests that reflect motor ability. Differences in grip-strength (ESLIM_009) were seen across all centers with J being higher than N, but the parameters affected were different, with some centers reporting differences in forelimb grip-strength and some for forelimb and hindlimb grip-strength combined (Figure ). Rotarod testing (ESLIM_010) showed significant differences in latency to fall across all centers, although the reduced motor ability of N was only seen for females in two of the centers. We further explored motor abilities in N and J male mice by examining motor learning performance on the rotarod over 4 days (Figure ). Whereas the motor performance of J mice improved markedly from day 1 to day 2, the performance of N mice improved only gradually, and was significantly different from the day 1 measurements only from day 3 ( P < 0.05) onwards. Moreover, fr...
Neurological, behavioral, and sensory
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).
Neurological, behavioral, and sensory
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.
Immune function and allergy
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 × 10 4 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 × 10 4 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...
Immune function and allergy
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....
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In parallel to the genomic analyses, the European Mouse Disease Clinic (EUMODIC) consortium has carried out a comprehensive phenotypic comparison of the C57BL/6NTac and C57BL/6J strains. EUMODIC comprises four mouse centers [ ] carrying out broad-based primary phenotyping of 500 mouse mutant knockout lines generated from the European Conditional Mouse Mutagenesis (EUCOMM) and Knockout Mouse (KOMP) projects within the IKMC program. Cohorts of mice from each mutant line enter the E uropean M ouse P henotyping Re source of S tandardised S creens slim (EMPReSSslim) phenotype assessment, which consists of two phenotyping pipelines, together comprising 20 phenotyping platforms (identified by an ESLIM__procedure_number) that are carried out from 9 to 15 weeks (see Additional file, Figure S1). The methods for performing each screen are detailed in the standard operating procedures (SOPs) that can be found in the EMPReSS database [ ]. Data were acquired on 413 phenotype parameters along with 146 metadata parameters, and entered into the EuroPhenome database [ ]. As part of this work, we have been capturing extensive control data on the baseline phenotype of C57BL/6NTac. We have also tak...
We conducted a number of tests that reflect motor ability. Differences in grip-strength (ESLIM_009) were seen across all centers with J being higher than N, but the parameters affected were different, with some centers reporting differences in forelimb grip-strength and some for forelimb and hindlimb grip-strength combined (Figure ). Rotarod testing (ESLIM_010) showed significant differences in latency to fall across all centers, although the reduced motor ability of N was only seen for females in two of the centers. We further explored motor abilities in N and J male mice by examining motor learning performance on the rotarod over 4 days (Figure ). Whereas the motor performance of J mice improved markedly from day 1 to day 2, the performance of N mice improved only gradually, and was significantly different from the day 1 measurements only from day 3 ( P < 0.05) onwards. Moreover, from day 2 to day 4 there were highly significant differences in the latency to fall between N and J. The primary testing carried out at the centers thus uncovered a potential reduced motor performance in N that was confirmed and further elaborated by more sophisticated testing of motor learning perfo...
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).
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.
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 × 10 4 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 × 10 4 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.
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).
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