Genetic analysis of strains C57BL/6J and BALB/cJ for Ath-1, a gene determining atherosclerosis susceptibility in mice

We previously reported that mice have at least one major gene determining atherosclerosis susceptibility, Ath-1. Susceptible alleles of Ath-1 are found in strain C57BL/6J and are associated with relatively low levels of high-density lipoprotein cholesterol (HDL-C) when these mice are fed an atherogenic diet. Resistant alleles of Ath-1 are found in strains C3H/HeJ and BALB/cJ and are associated with relatively high levels of HDL-C. Data reported earlier from the set of seven recombinant inbred (RI) strains, derived from C57BL/6By and BALB/cBy, showed that these parental strains differed at Ath-1. However, due to the limited number of RI strains, it was not possible to determine with certainty whether Ath-1 was the only major gene determining atherosclerosis susceptibility in these two strains or to determine its map position accurately. In this report, examination of F1, F2, and backcross progeny from a cross between C57BL/6J and BALB/cJ demonstrates that Ath-1 is the major gene determining atherosclerotic lesion formation and HDL-C levels in female mice. The data from male animals suggest that environmental factors or modifying genes also influence male HDL-C levels and thus partly obscure the Ath-1 phenotype. HDL-C levels in F1 progeny resemble the BALB/c parent. The data from the cross provide confirmatory evidence that Ath-1 is linked to Alp-2 on chromosome 1 with a map distance of 4.8 +/- 2.3 (SE). Combining these data with a previous cross between strain C57BL/6 and strain C3H/HeJ gives a map distance between Ath-1 and Alp-2 of 4.9 +/- 1.8 based on 7 crossovers found among 144 tested chromosomes.     

Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J.

Mice from the inbred strain C57BLKS/J (BKS) exhibit increased susceptibility to both diabetes and atherosclerosis compared to C57BL/6J (B6) mice. To determine whether the differences in diabetes and atherosclerosis are related, we carried out a cross between B6-db/db and BKS. We selected 99 female F2-db/db progeny, tested the progeny for plasma lipids, plasma glucose, and fatty-streak lesions, and used quantitative trait loci (QTL) analysis to identify the chromosomal regions associated with these phenotypes. No major QTL were found for total cholesterol, VLDL-cholesterol, or triglycerides. Two suggestive QTL were found for HDL-cholesterol (LOD scores of 2. 7 and 2.8), and two suggestive loci were found for plasma glucose (LOD scores of 2.3 and 2.0). Lesion size was not correlated with plasma lipid levels or glucose. Lesion size was determined by a locus at D12Mit49 with a LOD score of 2.5 and a significant likelihood ratio statistic. The gene for apolipoprotein apoB lies within the region, but apoB levels were similar in strains B6 and BKS. The QTL on Chr 12 was confirmed by constructing a congenic strain with BKS alleles in the QTL region on a B6 genetic background. We conclude that susceptibilities to diabetes and atherosclerosis are not conferred by the same genes in these strains and that a major gene on Chr 12, which we name Ath6, determines the difference in atherosclerosis susceptibility.     

Quantitative trait loci for tibial bone strength in C57BL/6J and C3H/HeJ inbred strains of mice

Three-point bending technology has been widely used in the measurement of bone strength. Quantitative trait loci (QTLs) for bone strength have been identified using mouse femurs. In this study, we investigate the use of mouse tibiae in identification of QTLs that regulate bone strength. Mouse tibiae were from a F₂ population derived from C57BL/6J (B6) and C3H/HeJ (C3H). Three-point bending was measured using ISO 4049, with the support width adjustable to accommodate specimen sizes outside the scope of ISO 4049. The strain rate is selectable from 0.05 to 500 mm per min. All stress strain diagrams are recorded and retrieved in digital electronic form. Genome scan was performed in The Jackson Laboratory (TJL). QTL mapping was conducted using Map Manager QTX software. Data show that (i) both elastic modulus (stiffness) and maximum loading (strength) value appear as normal distributions, suggesting that multiple genetic factors control the bone strength; (ii) 11 QTLs, accounting for 90% of variation for strength, have been detected. More than half QTLs of three-point bending are located on the same locations of bone density earlier identified from mouse femurs; (iii) a major QTL of femoral and vertebral bone mineral density (BMD) was not detected for bone strength of tibiae; (iv) the QTL on chromosome 4 has extremely high LOD score of 31.8 and represents 60% of the variation of bone strength; and (v) four QTLs of stiffness (chromosomes 2, 11, 15 and 19) have been identified.     

Circadian rhythm variation in activity, body temperature, and heart rate between C3H/HeJ and C57BL/6J inbred strains.

Inbred mice have been routinely used in studies of genetic effects that determine behavioral variation due to circadian rhythm. In addition to activity patterns (Act), we aimed to characterize variations in the circadian rhythm of deep-body temperature (T(db)) and heart rate (HR) in a specific genetic model of differential cardiorespiratory control. Radiotelemeters were implanted in C3H/HeJ (C3; n = 11) and C57BL/6J (B6; n = 11) inbred strains. Reciprocal first-generation offspring, B6C3F1/J (B6F1; n = 8) and C3B6F1 (C3F1; n = 3) mice, were included to initiate an evaluation of heritable phenotypes. Mice were housed individually in a facility maintained at 23-24 degrees C, and the light-dark cycle was set at 12-h intervals. In each animal, repeated measurements were obtained at 30-min intervals, and the circadian patterns of Act, T(db), and HR were assessed by novel statistical methods that detailed the periodic function for each strain. During the dark phase, B6 mice demonstrated two distinct peaks in Act and T(db) relative to a single early peak for C3 mice. In contrast to the parental strains, B6F1 and C3F1 mice demonstrated intermediate second peaks in Act and T(db). With respect to HR, the C3 strain demonstrated a significantly (P < 0.01) greater daily average compared with B6 mice. The circadian rhythm in HR differed significantly from the Act and T(db) patterns in B6 mice (but not in C3 mice); that is, the periodicity in HR for B6 mice preceded the rise and fall in Act and T(db) during both peaks. The B6 phenotype was also observed in F1 mice. In conclusion, these data suggest that the circadian regulation of Act, T(db), and HR vary significantly among C3, B6, and F1 mice. Furthermore, phenotypic differences between C3 and B6 strains can be used to explore the genetic basis for differential circadian regulation of body temperature and HR.     

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

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.     

Comparison of postnatal lung growth and development between C3H/HeJ and C57BL/6J mice.

Previous work by our group has demonstrated substantial differences in lung volume and morphometric parameters between inbred mice. Specifically, adult C3H/HeJ (C3) have a 50% larger lung volume and 30% greater mean linear intercept than C57BL/6J (B6) mice. Although much of lung development occurs postnatally in rodents, it is uncertain at what age the differences between these strains become manifest. In this study, we performed quasi-static pressure-volume curves and morphometric analysis on neonatal mice. Lungs from anesthetized mice were degassed in vivo using absorption of 100% O2. Pressure-volume curves were then recorded in situ. The lungs were then fixed by instillation of Zenker's solution at a constant transpulmonary pressure. The left lung from each animal was used for morphometric determination of mean air space chord length (Lma). We found that the lung volume of C3 mice was substantially greater than that of B6 mice at all ages. In contrast, there was no difference in Lma (62.7 microm in C3 and 58.5 microm in B6) of 3-day-old mice. With increasing age (8 days), there was a progressive decrease in the Lma of both strains, with the magnitude of the decrease in B6 Lma mice exceeding that of C3. C3 lung volume remained 50% larger. The combination of parenchymal architectural similarity with lung air volume differences and different rates of alveolar septation support the hypothesis that lung volume and alveolar dimensions are independently regulated.     

Lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in C57BL/J6 mice fed a high-fat diet

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.     

Chromosomal loci that influence oral nicotine consumption in C57BL/6J x C3H/HeJ F2 intercross mice

Several studies have demonstrated that there are genetic influences on free-choice oral nicotine consumption in mice. In order to establish the genetic architecture that underlies individual differences in free-choice nicotine consumption, quantitative trait loci (QTL) mapping was used to identify chromosomal regions that influence free-choice nicotine consumption in male and female F(2) mice derived from a cross between C57BL/6J and C3H/HeJ mice. These two mouse strains were chosen not only because they differ significantly for oral nicotine consumption, but also because they are at or near phenotypic extremes for all measures of nicotine sensitivity that have been reported. A four-bottle choice paradigm was used to assess nicotine consumption over an 8-day period. The four bottles contained water or water supplemented with 25, 50 or 100 microg/ml of nicotine base. Using micrograms of nicotine consumed per milliliter of total fluid consumed per day as the nicotine consumption phenotype, four significant QTL were identified. The QTL with the largest LOD score was located on distal chromosome 1 (peak LOD score = 15.7). Other chromosomes with significant QTL include central chromosome 4 (peak LOD score = 4.1), proximal chromosome 7 (peak LOD score = 6.1) and distal chromosome 15 (peak LOD score = 4.8). These four QTL appear to be responsible for up to 62% of the phenotypic variance in oral nicotine consumption.     

Differential Effects of Fluoride During Osteoblasts Mineralization in C57BL/6J and C3H/HeJ Inbred Strains of Mice

The behavior of fluoride ions in biological systems has advantages and problems. On one hand, fluoride could be a mitogenic stimulus for osteoblasts. However, high concentrations of this element can cause apoptosis in rat and mouse osteoblasts. Toward an understanding of this effect, we examined the role of sodium fluoride (NaF) in two mouse calvaria osteoblasts during the mineralization process. The animals used were C3H/HeJ (C3) and C57BL/6J (B6) mice. The calvaria cells were cultured for 28 days in the presence of several doses of NaF (0, 5, 10, 25, 50, and 75 μM), and we performed the assays: mineralized nodule measurements, alkaline phosphatase (ALP) activity, determination of type I collagen, and matrix metalloproteinase-2 (MMP-2) activity. The results showed no effects on alkaline phosphatase activity but decreased mineralized nodule formation. In B6 cells, the NaF effect was already seen with 10 μM of NaF and a greater increase of cellular type I collagen, and MMP-2 activity was upregulated after 7 days of NaF exposure. C3 osteoblasts showed a reduction in the mineralization pattern only after 50 μM of NaF with a slight increase of type I collagen and downregulation of MMP-2 activity during the mineralization period. In conclusion, fluoride affects the production and degradation of the extracellular matrix during early onset and probably during the mineralization period. Additionally, the genetic factors may contribute to the variation in cell response to fluoride exposure, and the differences observed between the two strains could be explained by an alteration of the bone matrix metabolism (synthesis and degradation).     

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response‐related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan‐induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)‐γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN‐γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN‐γ doses (0.2 μg/kg) either alone or in combination with the ß‐glucan‐binding CD5 protein (0.7 mg/kg) leading to improved post zymosan‐induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10⁴ CFU/gr) were ameliorated by low‐dose IFN‐γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.     

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.     

Genetic differentiation within the inbred C57BL/6J mouse strain

The C57BL/6J (B6) inbred mouse strain is commonly used in biomedical researches. However, some unexpected inconsistency was reported compared with previous studies, and in most cases, it can be attributed to environmental, epigenetic or stochastic differences. The goal of this study was to investigate the genetic stability of the B6 strain maintained in different breeders. B6 mice purchased from five Chinese commercial breeders were examined, and mitochondrial D-loop sequence and 18 microsatellite loci were genotyped. There is no difference in the D-loop sequences, but variations exist in the nucleic microsatellite markers. Combining the data from MGI_4.01, a significant divergence is observed among those mice. The present study indicates that different B6 mice share the common maternal lineage and are still inbred in each breeder, but subline divergence occurs.     

Genetic analysis of alcohol intake in recombinant inbred and congenic strains derived from A/J and C57BL/6J progenitors

The objective of the present study was to map quantitative trait loci (QTL) for alcohol intake using A × B/B × A recombinant inbred (RI) and AcB/BcA recombinant congenic (RC) strains of mice that were independently derived from the A/J and C57BL/6J progenitors. Mice were screened for levels of alcohol consumption with four days of forced exposure to alcohol, followed by three weeks of free choice between water and a 10% alcohol solution. Alcohol consumption data previously collected for 27 A × B/B × A RI strains were reanalyzed using a larger marker set and composite interval mapping. The reanalysis found markers on Chromosome 2 (D2Mit74, 107 cM) (males and females) and on Chromosome 11 (Pmv22, 8 cM) (females only) that exceeded the threshold for significant loci, and found suggestive loci (in males) on Chromosomes 10 (D10 Mit126, 21 cM), 12 (D12Mit37, 1 cM), 15 (Pdgfb, 46.8 cM), and 16 (D16Mit125, 29 cM). An additional suggestive locus was identified in female RI mice on Chromosome 11 (D11Mit120, 47.5 cM). Composite interval mapping (CIM) analysis indicated that there was a significant association between loci at Pdgfb and D2Mit74 in both males and females. Analysis of the AcB/BcA RC strains identified 11 QTL on Chromosomes 2, 3, 5,6, 7, 8, 9, 10, 12, 13, and 15. QTL on Chromosomes 7, 10, 12, and 15 were identified in both the A × B/B × A RI and AcB/BcA RC strains of mice. Additional QTLs identified on Chromosomes 2, 3, 7, 11, and 15 overlap with those previously identified in the literature using strains of mice with a C57BL/6J progenitor.     

Macrophage cholesterol efflux to free apoprotein A-I in C3H and C57BL/6 mice

Cholesterol efflux from peritoneal macrophages of mice C57BL/6 susceptible and C3H resistant to atherosclerosis was compared, using apoprotein A-I as acceptor. The elicited macrophages were labeled with 3H-cholesterol and cholesterol enriched by incubation for 24 h with acetylated LDL. After incubation for 6 or 24 h, 3H-cholesterol efflux to free apoA-I (10 microg/ml) was significantly higher with macrophages derived from C3H mice compared to C57BL/6 mice. The cells were also pretreated with 0.3-0.45 mM cyclic AMP, 10 microM 9-cis-retinoic acid or 10 microM 22(R)-hydroxycholesterol, RXR and LXR ligands. Treatment with cyclic AMP, RXR, or LXR ligands, resulted in enhancement of 3H-cholesterol efflux in both strains. Under all conditions, 3H-cholesterol efflux was significantly higher in C3H compared to C57BL/6 macrophages. In conclusion, the higher cholesterol efflux from C3H macrophages could contribute toward the resistance of this strain to diet-induced atherosclerosis despite hypercholesterolemia.     

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

Background

Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice.

Methodology/Principal Findings

We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1–2 months, 3–8 months and 9–12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5^th and 97.5^th percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05–0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K⁺, Ca⁺⁺, PO₄ ⁻ (p<0.05) and for TAG, Chol, AST, Fe⁺⁺ (p<0.001) in 4–8 month-old animals; (2) for CK, Crea, Mg⁺⁺, Na⁺⁺, K⁺, Cl⁻ (p<0.05) and BUN (p<0.001) in 2- and in 10–12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span.

Conclusion/Significance

Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.     

Streptobacillus moniliformis epizootic in barrier-maintained C57BL/6J mice and susceptibility to infection of different strains of mice

We report a Streptobacillus moniliformis epizootic in barrier-maintained SPF mice. Although various inbred and F1 hybrid strains of mice have been kept in this animal facility, only C57BL/6J Han [corrected] mice showed clinical signs of disease. During the course of the epizootic, 825 breeding animals (approximately 36% of the breeders) died or had to be killed because of severe clinical signs. Although sequential treatment with ampicillin and chlortetracycline gave good therapeutic results, the animal facility was vacated in order to exclude any risk of cross-contamination of the other rodent colonies in our institute. The source and route of transmission of S. moniliformis could not be elucidated. To investigate strain dependent differences experimental infection of different strains of mice with our S. moniliformis isolate was performed. After oral infection only C57BL/6J showed the typical signs of a cervical lymphadenitis and gave an immunological response. BALB/cJ, C3H/He, DBA/2J, CB6F1 and B6D2F1 mice were not affected except in two cases of DBA/2J and B6D2F1 mice where seroconversion was observed. After intravenous infection of C57BL/6J, DBA/2J [corrected] and BALB/cJ all animals showed positive titers in the indirect immunofluorescence test (IIF). One hundred percent of the C57BL/6J, forty percent of the DBA/2J, and none of the BALB/cJ mice developed severe symptoms. The results demonstrate that the susceptibility to streptobacillosis is predominantly influenced by genetic factors.     

Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.