Systematic analysis of emotionality in consomic mouse strains established from C57BL/6J and wild-derived MSM/Ms

Consomic strains have recently attracted attention as an advantageous method to screen for genes related to developmental, physiological, and behavioral phenotypes. Recently, a new set of consomic strains was established from the Japanese wild-derived mouse strain MSM/Ms and C57BL/6JJcl. By analyzing the entire consomic panel, we were able to identify a number of chromosomes associated with anxiety-like behaviors in the open-field (OF) test, a light-dark box and an elevated plus maze. Detailed observation of the OF behavior allowed us to identify chromosomes associated with those ethological traits, such as stretch attend, rearing, and jumping. Repeated OF test trials have different meanings for animals, and we found that some chromosomes responded to only the first or second trial, while others were consistent across both trials. By examining both male and female mice, sex-dependent effects were found in several measurements. Principal component analysis of anxiety-like behaviors extracted five factors: 'general locomotor activity', 'thigmotaxis', 'risk assessment', 'open-arm exploration' and 'autonomic emotionality'. We mapped chromosomes associated with these five factors of emotionality.     

Segregation of a QTL cluster for home-cage activity using a new mapping method based on regression analysis of congenic mouse strains

Recent genetic studies have shown that genetic loci with significant effects in whole-genome quantitative trait loci (QTL) analyses were lost or weakened in congenic strains. Characterisation of the genetic basis of this attenuated QTL effect is important to our understanding of the genetic mechanisms of complex traits. We previously found that a consomic strain, B6-Chr6C^MSM, which carries chromosome 6 of a wild-derived strain MSM/Ms on the genetic background of C57BL/6J, exhibited lower home-cage activity than C57BL/6J. In the present study, we conducted a composite interval QTL analysis using the F2 mice derived from a cross between C57BL/6J and B6-Chr6C^MSM. We found one QTL peak that spans 17.6 Mbp of chromosome 6. A subconsomic strain that covers the entire QTL region also showed lower home-cage activity at the same level as the consomic strain. We developed 15 congenic strains, each of which carries a shorter MSM/Ms-derived chromosomal segment from the subconsomic strain. Given that the results of home-cage activity tests on the congenic strains cannot be explained by a simple single-gene model, we applied regression analysis to segregate the multiple genetic loci. The results revealed three loci (loci 1–3) that have the effect of reducing home-cage activity and one locus (locus 4) that increases activity. We also found that the combination of loci 3 and 4 cancels out the effects of the congenic strains, which indicates the existence of a genetic mechanism related to the loss of QTLs.     

QTL analyses of spontaneous activity by using mouse strains from Mishima battery

We reported previously that spontaneous activity in the home cage is highly variable among the Mishima battery of mouse strains. In that study, NJL and KJR were found to be hyperactive strains in contrast to BLG2, which showed one of the lowest activity levels. To unravel the genetic loci involved in this behavioral phenotype, we conducted QTL analyses on backcross populations of crosses between either NJL or KJR and BLG2 strains. In the backcross of NJL to BLG2, no single locus was associated with increased spontaneous activity. In the backcross of KJR to BLG2, linkage analysis showed that a locus on the most telomeric region of Chromosome (Chr) 3 was involved in the spontaneous activity, thus named Loco1. Further linkage analysis using selected progeny carrying the allele from KJR at the Loco1 locus suggested the presence of another locus, Loco2, on Chr 17. An analysis showed that Loco1 and Loco2 interacted epistatically.     

Multigenic factors associated with a hydrocephalus-like phenotype found in inter-subspecific consomic mouse strains

Hydrocephalus is a significant clinical condition in humans and is known to be a multifactorial neurologic disorder. It has been thought that genetic factors are closely involved in the etiology of congenital hydrocephalus, but further investigation is required to elucidate the genetic architecture of hydrocephalus. By analyzing breeding records of a panel of inter-subspecific consomic mouse strains, we found that consomic strains with MSM/Ms (MSM) chromosomes 4, 5, 7, 11, 15, and 17 showed a significantly higher incidence of hydrocephalus, whereas both parental strains, MSM and C57BL/6J (B6), rarely showed this abnormality. Further analysis of the consomic Chr 17 strain revealed that apparently normal individuals of this strain also exhibited increased brain ventricle size compared to B6 and had larger individual variation of ventricle size within the strain. Thus, we concluded that hydrocephalus is an extreme phenotype of individual ventricle size variation. We then established and analyzed several subconsomic strains of Chr 17 to identify genetic factors related to hydrocephalus-like phenotype and successfully mapped one genetic locus around the proximal region of Chr 17.     

Genetic determinants on rat chromosome 6 modulate variation in the hypercapnic ventilatory response using consomic strains.

To understand the genetic basis of pathways involved in the control of breathing, a large scale, high-throughput study using chromosomal substitution strains of rats is underway. Eight new consomic rat stains (SS-2(BN), SS-4(BN), SS-6(BN), SS-7(BN), SS-8(BN), SS-11(BN), SS-12(BN), SS-14(BN), SS-Y(BN)), containing one homozygous BN/NHsdMcwi (BN) chromosome on a background of SS/JrHsdMcwi (SS), were created by PhysGen (http://pga.mcw.edu) Program for Genomic Applications. Male and female rats were studied using standard plethysmography under control conditions and during acute hypoxia (inspired oxygen fraction = 0.12) and hypercapnia (inspired CO(2) fraction = 0.07). The rats were also studied during treadmill exercise. Both male and female BN rats had a significantly lower ventilatory response during 7% CO(2) compared with SS rats of the same gender. SS-6(BN) female rats had a significantly reduced ventilatory response, similar to BN rats due primarily to a reduced tidal volume. Male SS-6(BN) rats had a significantly reduced tidal volume response to hypercapnia but a slightly increased frequency response during hypercapnia. Gene(s) on the Y chromosome may play a role in this increased frequency response in the male rats because the SS-Y(BN) hypercapnic ventilatory response involves a significantly increased frequency response. Several chromosomal substitutions slightly altered the ventilatory responses to hypoxia and exercise. However, genes on chromosomes 6 and Y of those studied are of primary importance in aspects of ventilatory control currently studied.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J?×?A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J × A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.

     

Lith6: a new QTL for cholesterol gallstones from an intercross of CAST/Ei and DBA/2J inbred mouse strains

A complex genetic basis determines the individual predisposition to develop cholesterol gallstones in response to environmental factors. We employed quantitative trait locus/loci (QTL) analyses of an intercross between inbred strains CAST/Ei (susceptible) and DBA/2J (resistant) to determine the subset of gallstone susceptibility (Lith) genes these strains possess. Parental and first filial generation mice of both genders and male intercross offspring were evaluated for gallstone formation after feeding a lithogenic diet. Linkage analysis was performed using a form of multiple interval mapping. One significant QTL colocalized with Lith1 [chromosome (chr) 2, 50 cM], a locus identified previously. Significantly, new QTL were detected and named Lith10 (chr 6, 4 cM), Lith6 (chr 6, 54 cM), and Lith11 (chr 8, 58 cM). Statistical and genetic analyses suggest that Lith6 comprises two QTL in close proximity. Our molecular and genetic data support the candidacy of peroxisome proliferator-activated receptor gamma (Pparg) and Slc21a1, encoding Pparg, and the basolateral bile acid transporter SLC21A1 (Slc21a1/Oatp1), respectively, as genes underlying Lith6.     

A marker assisted selection protocol (MASP) to generate C57BL/6J or 129S6/SvEvTac speed congenic or consomic strains

A marker assisted selection protocol is presented that allows for the generation of congenic or consomic strains derived from a C57BL/6J:129S6/SvEvTac mixed strain background. The protocol uses defined primer pairs to generate amplicons that can be distinguished by non-denaturing agarose electrophoresis. Use of this application should result in substantial savings in time, effort, and cost for investigators in all areas of transgenic mouse research.     

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.     

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina^® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ^c?2j /J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.     

Xanthine oxidase activity and immunologically detectable protein in the C57B1/6 mouse

1. Xanthine oxidase (XO) was purified from livers of C57B1/6 mice. Antibodies generated against the purified protein were used in an immunoassay to measure total XO protein. 2. Both the specific activity and amount of XO protein were greater in the proximal small intestine than in the liver. A pool of inactive enzyme was present in the small intestine which developed after weaning. 3. Male C57B1/6 mice had the same XO specific activity as females and neither the hepatic nor the intestinal XO activity were affected by the level of dietary protein. 4. When mice were fed diets with tungsten, XO activity was lost and the amount of XO protein in the small intestine was decreased.     

Comparison of intestine and bone marrow radiosensitivity of the BALB/c and the C57BL/6 mouse strains and their B6CF1 offspring

The radiosensitivity as measured by LD50/6 or LD50/30 of the F1 hybrid B6CF1 (C57BL/6 X BALB/c) is similar to that of C57BL/6 mice but markedly different from BALB/c. The LD50/6 for BALB/c mice was about 8.8 Gy compared to 16.4 Gy for the B6CF1. The difference in LD50/6 between the parent strains or between BALB/c and the F1 hybrid could not be explained by any differences in crypt cell number, cell cycle time, or transit time. Likewise, the observed differences in the LD50/6 do not appear to result from marked differences in the radiosensitivity of marrow stem cells (CFU-S) since the D0's for the three genotypes of mice were similar. Also, there were no apparent differences in the red blood cell contents of several enzymes associated with antioxidant defenses. The microcolony assay was used to determine the D0 for the crypt clonogenic cells and the D0 values for 60Co gamma rays were about 0.8 Gy for BALB/c mice and 1.4 Gy for B6CF1 mice. However, the D0 values for JANUS fission neutrons were similar; 0.6 Gy for the BALB/c mice and 0.5 for the B6CF1 mice. A comparison of clonogenic cell kinetics, using prolonged colcemid block to distinguish between slowly and rapidly cycling cells suggest that, normally, the stem cells are slowly cycling in both the BALB/c and the B6CF1 hybrid. However, the stem cells of the B6CF1 appear to go into rapid cell cycle more rapidly than those of the BALB/c following irradiation or prolonged colcemid treatment. The more rapid recovery in intestinal epihelial cell production in the B6CF1 hybrid after irradiation may provide an increased mucosal barrier and may, in part, explain the difference in the response to radiation compared to that in the BALB/c.     

Parallel inheritance of tissue catalase activity and immunostimulatory action of amphotericin B in inbred mouse strains

Both amphotericin B (AmB) and its methyl ester derivative are potent immunoadjuvants that also stimulate murine B lymphocytes and macrophages in vitro. Most of the common inbred mouse strains show AmB-induced immunostimulation (AmB-high responders) but mice from the C57BL strains, regardless of H-2 genotype, are AmB-low responders. Lymphoid cells from AmB-high responder strains also exhibit greater resistance to H2O2 toxicity in vitro compared with cells from AmB-low responders. This result led to an evaluation of differences in the tissue catalase levels of AmB-high and -low responder strains. Results from several laboratories, including ours, indicate that C57BL mouse strains express low levels of tissue catalase activity in addition to low or absent immunostimulant effects of AmB. Several AmB-high responder strains have high spleen cell, macrophage, and liver catalase, and the mouse strain distribution of enzyme activity as well as the dominant inheritance of the low catalase phenotype is compatible with regulation by the Ce-1 locus in lymphoid organs as well as liver. Other evidence also suggests that H2O2 metabolism is important in lymphoid cell responses to AmB. For example, AmB stimulates a stronger respiratory burst in macrophages from AmB-high responder strains under the same conditions that inhibit burst activity in macrophages from low responders. Selective immune enhancement by AmB in high catalase mouse strains along with enhanced susceptibility to AmB toxicity in low responder C57BL mice with low tissue catalase activity suggests that cellular peroxidation is a major determinant of the genetic regulation of amphotericin-induced immunostimulation.     

Dissociation between macrophage tumoricidal capacity and suppressive activity: analysis with macrophage-defective mouse strains

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.     

Genetic analysis of mortality in murine angiostrongyliasis costaricensis using SMXA recombinant inbred mouse strains

The SMXA recombinant inbred mouse strain set was produced by systematic inbreeding from the F2 generation of a cross between two progenitor inbred strains, A/J and SM/J, which differed markedly with respect to the patterns of infection with Angiostrongylus costaricensis. We have applied this set to genetic analysis of mouse susceptibility to this nematode infection. The mortality was variable among substrains of the SMXA RI strains, indicating the involvement of multiple genes. Linkage analysis showed several chromosomal regions closely linked to mortality; chromosome 6 (D6Rik86, 87; P0.001), 10 (D10Rik66–D10Mit12; P=0.0058), 13 (D13Rik79, 80; P=0.0096) and 17 (D17Mit28–D17Rik76; P=0.0088).     

QTL mapping for genetic determinants of lipoprotein cholesterol levels in combined crosses of inbred mouse strains

To identify additional loci that influence lipoprotein cholesterol levels, we performed quantitative trait locus (QTL) mapping in offspring of PERA/EiJxI/LnJ and PERA/EiJxDBA/2J intercrosses and in a combined data set from both crosses after 8 weeks of consumption of a high fat-diet. Most QTLs identified were concordant with homologous chromosomal regions that were associated with lipoprotein levels in human studies. We detected significant new loci for HDL cholesterol levels on chromosome (Chr) 5 (Hdlq34) and for non-HDL cholesterol levels on Chrs 15 (Nhdlq9) and 16 (Nhdlq10). In addition, the analysis of combined data sets identified a QTL for HDL cholesterol on Chr 17 that was shared between both crosses; lower HDL cholesterol levels were conferred by strain PERA. This QTL colocalized with a shared QTL for cholesterol gallstone formation detected in the same crosses. Haplotype analysis narrowed this QTL, and sequencing of the candidate genes Abcg5 and Abcg8 confirmed shared alleles in strains I/LnJ and DBA/2J that differed from the alleles in strain PERA/EiJ. In conclusion, our analysis furthers the knowledge of genetic determinants of lipoprotein cholesterol levels in inbred mice and substantiates the hypothesis that polymorphisms of Abcg5/Abcg8 contribute to individual variation in both plasma HDL cholesterol levels and susceptibility to cholesterol gallstone formation.