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.     

Regulation of the expression of two female-predominant CYP3A mRNAs (CYP3A41 and CYP3A44) in mouse liver by sex and growth hormones

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs.     

Cholesterol 25-hydroxylation activity of CYP3A

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.     

Amelioration of PXR-mediated CYP3A4 induction by mGluR2 modulators

This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.     

Transglutaminase 3 expression in C57BL／6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.     

Substrain specific behavioral responses in male C57BL/6N and C57BL/6J mice to a shortened 21-hour day and high-fat diet

ABSTRACT

Altered circadian rhythms have negative consequences on health and behavior. Emerging evidence suggests genetics influences the physiological and behavioral responses to circadian disruption. We investigated the effects of a 21 h day (T = 21 cycle), with high-fat diet consumption, on locomotor activity, explorative behaviors, and health in male C57BL/6J and C57BL/6N mice. Mice were exposed to either a T = 24 or T = 21 cycle and given standard rodent chow (RC) or a 60% high-fat diet (HFD) followed by behavioral assays and physiological measures. We uncovered numerous strain differences within the behavioral and physiological assays, mainly that C57BL/6J mice exhibit reduced susceptibility to the obesogenic effects of (HFD) and anxiety-like behavior as well as increased circadian and novelty-induced locomotor activity compared to C57BL/6N mice. There were also substrain-specific differences in behavioral responses to the T = 21 cycle, including exploratory behaviors and circadian locomotor activity. Under the 21-h day, mice consuming RC displayed entrainment, while mice exposed to HFD exhibited a lengthening of activity rhythms. In the open-field and light-dark box, mice exposed to the T = 21 cycle had increased novelty-induced locomotor activity with no further effects of diet, suggesting daylength may affect mood-related behaviors. These results indicate that different circadian cycles impact metabolic and behavioral responses depending on genetic background, and despite circadian entrainment.     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

A model of sleep-disordered breathing in the C57BL/6J mouse.

To investigate the pathophysiological sequelae of sleep-disordered breathing (SDB), we have developed a mouse model in which hypoxia was induced during periods of sleep and was removed in response to arousal or wakefulness. An on-line sleep-wake detection system, based on the frequency and amplitude of electroencephalograph and electromyograph recordings, served to trigger intermittent hypoxia during periods of sleep. In adult male C57BL/6J mice (n = 5), the sleep-wake detection system accurately assessed wakefulness (97.2 +/- 1.1%), non-rapid eye movement (NREM) sleep (96.0 +/- 0.9%) and rapid eye movement (REM) sleep (85.6 +/- 5.0%). After 5 consecutive days of SDB, 554 +/- 29 (SE) hypoxic events were recorded over a 24-h period at a rate of 63.6 +/- 2.6 events/h of sleep and with a duration of 28.2 +/- 0.7 s. The mean nadir of fraction of inspired O(2) (FI(O(2))) on day 5 was 13.2 +/- 0.1%, and 137.1 +/- 13.2 of the events had a nadir FI(O(2)) <10% O(2). Arterial blood gases confirmed that hypoxia of this magnitude lead to a significant degree of hypoxemia. Furthermore, 5 days of SDB were associated with decreases in both NREM and REM sleep during the light phase compared with the 24-h postintervention period. We conclude that our murine model of SDB mimics the rate and magnitude of sleep-induced hypoxia, sleep fragmentation, and reduction in total sleep time found in patients with moderate to severe SDB in the clinical setting.     

Discriminating activation of CYP2B9 expression in male C57BL/6 mouse liver by beta-estradiol

The inducible expression of the cytochrome P450 2B subfamily was investigated in male C57BL/6 (B6) and DBA/2 (D2) mice, as well as their hybrids, B6D2F1, at the mRNA level. The expression of hepatic CYP2B mRNAs in B6 was lightly induced by beta-estradiol (ES), while that by phenobarbital (PB) or 1,1,1-trichloro-2, 2-bis(p-chlorophenyl) ethane (DDT) was prominent. Discriminating analysis showed a novelty that ES markedly induced CYP2B9 mRNA expression, whereas PB and DDT increased CYP2B10 more than CYP2B9 expression: albeit both mRNA species responded to all three inducers. Furthermore, the specific induction by ES of CYP2B9 mRNA in B6 male mice, but not D2 male mice, suggests strain dependency in the regulatory pathway of CYP2B9 expression.     

7-nitroindazole and posthypoxic ventilatory behavior in the A/J and C57BL/6J mouse strains.

Periodic breathing (PB) is a fundamental breathing pattern in many common cardiopulmonary illnesses. The finding of PB in C57BL/6J (B6) mice was previously ascribed to strain differences in posthypoxic ventilatory and frequency decline in the B6 mice (Han F, Subramanian S, Price ER, Nadeau J, and Strohl KP. J Appl Physiol 92: 1133-1140, 2002). We tested whether the induction of posthypoxic frequency decline in A/J mice, through administration of a neuronal nitric oxide synthase blocker [7-nitroindazole (7-NI); 60 mg/kg], would cause A/J mice to exhibit PB and/or alter PB expression in the B6 strain. Recordings of ventilatory behavior by the plethysmography method were made when unanesthetized B6 (n = 10) or A/J (n = 6) animals were reoxygenated with 100% O2 or room air after exposure to 8% O2. Before undergoing gas challenges, mice were given an intraperitoneal injection of either peanut oil alone (vehicle) or 7-NI suspended in peanut oil. Compared with vehicle, both strains of mice exhibited posthypoxic frequency decline and the absence of short-term potentiation with 7-NI administration. B6 mice continued to exhibit posthypoxic PB; however, the PB was characterized by longer cycle and apnea length. In contrast, A/J mice did not show increased tendency toward posthypoxic PB with 7-NI. We conclude that 7-NI further differentiates the A/J and B6 strains in terms of PB and that strain-related differences in posthypoxic frequency decline are not primary determinants of this strain difference in the occurrence of PB. Metabolism was not associated with either the expression of posthypoxic ventilatory decline or PB. Furthermore, neuronal nitric oxide may be an organizing feature in the presence, length, and/or cycle length of apnea in the susceptible strain.     

Maternal control of seed size by EOD3/CYP78A6 in Arabidopsis thaliana

Seed size in higher plants is coordinately determined by the growth of the embryo, endosperm and maternal tissue, but relatively little is known about the genetic and molecular mechanisms that set final seed size. We have previously demonstrated that Arabidopsis DA1 acts maternally to control seed size, with the da1-1 mutant producing larger seeds than the wild type. Through an activation tagging screen for modifiers of da1-1, we have identified an enhancer of da1-1 (eod3-1D) in seed size. EOD3 encodes the Arabidopsis cytochrome P450/CYP78A6 and is expressed in most plant organs. Overexpression of EOD3 dramatically increases the seed size of wild-type plants, whereas eod3-ko loss-of-function mutants form small seeds. The disruption of CYP78A9, the most closely related family member, synergistically enhances the seed size phenotype of eod3-ko mutants, indicating that EOD3 functions redundantly with CYP78A9 to affect seed growth. Reciprocal cross experiments show that EOD3 acts maternally to promote seed growth. eod3-ko cyp78a9-ko double mutants have smaller cells in the maternal integuments of developing seeds, whereas eod3-1D forms more and larger cells in the integuments. Genetic analyses suggest that EOD3 functions independently of maternal factors DA1 and TTG2 to influence seed growth. Collectively, our findings identify EOD3 as a factor of seed size control, and give insight into how plants control their seed size.     

Effects of buspirone on posthypoxic ventilatory behavior in the C57BL/6J and A/J mouse strains.

Buspirone, a partial agonist of the serotonergic 5-HT1A receptor, improves breathing irregularities in humans with Rett syndrome or brain stem injury. The purpose of this study was to examine whether buspirone alters posthypoxic ventilatory behavior in C57BL/6J (B6) and A/J mouse strains. Measurements of ventilatory behavior were collected from unanesthetized adult male mice (n=6 for each strain) using the plethysmographic method. Mice were given intraperitoneal injections of vehicle or several doses of buspirone and exposed to 2 min of hypoxia (10% O2) followed by rapid reoxygenation (100% O2). Twenty minutes later, mice were tested for hypercapnic response (8% CO(2)-92% O2). On a separate day, mice were injected with the 5-HT1A receptor antagonist 4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl] ethyl}-N-2-pyridinylbenzamide (p-MPPI) before the injection of buspirone, and measurements were repeated. In separate studies, arterial blood-gas analysis was performed for each strain (n=12 in B6 and 10 in A/J) with buspirone or vehicle. In both strains, buspirone stimulated ventilation at rest. In the B6 mice, the hypoxic response was unchanged, but the response to hypercapnia was reduced with buspirone (5 mg/kg; P<0.05). With reoxygenation, vehicle-treated B6 exhibited periodic breathing and greater variation in ventilation compared with A/J (P<0.01). In B6 animals, >or=3 mg/kg of buspirone reduced variation and prevented the occurrence of posthypoxic periodic breathing. Both effects were reversed by p-MPPI. Treatment effect of buspirone was not explained by a difference in resting arterial blood gases. We conclude that buspirone improves posthypoxic ventilatory irregularities in the B6 mouse through its agonist effects on the 5-HT1A receptor.     

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val(117)--> Ala and Arg(372)--> His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala(117) --> Val and His(372) --> Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases

We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3′:5′ cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca²⁺/calmodulin-dependent protein kinases II (independently of Ca²⁺) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria

The objective of this study was to characterize the lipidome and electron transport chain activities in purified non-synaptic (NS) and synaptic (Syn) mitochondria from C57BL/6J mouse cerebral cortex. Contamination from subcellular membranes, especially myelin, has hindered past attempts to accurately characterize the lipid composition of brain mitochondria. An improved Ficoll and sucrose discontinuous gradient method was employed that yielded highly enriched mitochondrial populations free of myelin contamination. The activities of Complexes I, II, III, and II/III were lower in Syn than in NS mitochondria, while Complexes I/III and IV activities were similar in both populations. Shotgun lipidomics showed that levels of cardiolipin (Ptd₂Gro) were lower, whereas levels of ceramide and phosphatidylserine were higher in Syn than in NS mitochondria. Coenzyme Q₉ and Q₁₀ was also lower in Syn than in NS mitochondria. Gangliosides, phosphatidic acid, sulfatides, and cerebrosides were undetectable in brain mitochondria. The distribution of Ptd₂Gro molecular species was similar in both populations and formed a unique pattern, consisting of seven major molecular species groups, when arranged according to mass to charge ratios. Remodeling involving choline and ethanolamine phosphoglycerides could explain Ptd₂Gro heterogeneity. NS and Syn mitochondrial lipidomic heterogeneity could influence energy metabolism, which may contribute to metabolic compartmentation of the brain.     

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.