Genetic Determination of the Developmental Program for Mouse Liver β-GALACTOSIDASE: Involvement of Sites Proximate to and Distant from the Structural Gene

The identification and mode of action of genetic loci that program gene expression during development are important for understanding differentiation in higher organisms. Previous work from this laboratory has identified two patterns for the postnatal development of liver beta-galactosidase among inbred mouse strains: type I, where activity levels remain constant after about 30 days of age, is found in strains DBA/2J, CBA/J, and BALB/cJ, among others; type II, where activity levels increase between 25 and 50 days of age to reach a new adult level, is found in strain C57BL/6J and related strains. It has been shown that the type I vs. type II developmental difference between strains C57BL/6J and DBA/2J is due to variation at a locus, Bgl-t, that maps with the beta-galactosidase complex, [Bgl], on chromosome 9. In the present study, we have confirmed the existence of Bgl-t as a temporal locus within [Bgl] by analysis of both a congenic strain carrying the beta-galactosidase complex of strain CBA/J in the C57BL/6J genetic background and a cross of strains CBA/J and C57BL/6J. The existence of additional temporal loci for beta-galactosidase that segregate independently of the structural gene and participate in determination of the type I vs. type II difference was revealed by analysis of: (1) a congenic strain containing the beta-galactosidase complex of strain BALB/cJ in the C57BL/10Sn background; (2) recombinant inbred lines derived from progenitor strains C57BL/6ByJ and BALB/cByJ; and (3) a genetic cross between strains C57BL/6ByJ and BALB/cByJ. Thus, for these pairs of strains, the type I vs. type II developmental difference is due to variation at a temporal locus (or loci) unlinked to the enzyme structural gene, and not at Bgl-t. These facts, together with information gathered from an examination of the distribution of beta-galactosidase phenotypes among over 100 inbred strains (Breen, Lusis and Paigen 1977), have led us to conclude that the postnatal developmental pattern for liver beta-galactosidase is determined by a set of interacting temporal genes. One of these, Bgl-t, is located within [Bgl], and one or more are separable from [Bgl] by recombination. A possible mode of interaction among the temporal and instructural loci is suggested.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Kinetics of ectromelia virus (mousepox) transmission and clinical response in C57BL/6j, BALB/cByj and AKR/J inbred mice

Research was undertaken to answer basic questions on susceptibility, clinical response and transmission of ectromelia virus in selected strains of inbred mice. C57BL/6J and AKR/J were found to be markedly more resistant to a virulent strain of ectromelia virus (isolated during the 1979-80 outbreak at the National Institutes of Health) than C57LJ, BALB/cByJ, DBA/2J, A.By/SNJ and C3H/HeJ when infected by footpad inoculation. In C57BL/6J and AKR/J the LD50 was about 7 logs higher than the ID50. With one exception, C57LJ, the LD50 and ID50 titers in the other strains were about equal. In C57LJ the LD50 titer was intermediate. Following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and up to 29 days from BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days respectively after infection. Virus was isolated from the spleen in 2 of 5 BALB/cByJ mice and 1 of 7 C57BL/6J mice tested 95 days after gastric inoculation. Following footpad inoculation, BALB/cByJ mice consistently transmitted virus to cage mates before death at 10-12 days. C57BL/6J mice transmitted between days 8 and 17, but not beyond. Virus was maintained in C57BL/6J mice by exposure to infected cage mates for seven passages, which was the most attempted. Clinical signs in infected C57BL/6J mice were usually subtle or inapparent.     

Lens density tracking in mice by Scheimpflug imaging

Scheimpflug imaging has recently been established for in vivo imaging of the anterior eye segment and quantitative determination of lens transparency in the mouse. This enables more effective investigations of cataract formation with the mouse model, including longitudinal studies. In order to enable recognition of disease-associated irregularities, we performed Scheimpflug measurements with the common laboratory inbred lines C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129/SvJ in a period between 2 and 12 months of age. C57BL/6J mice showed lowest mean lens densities during the test period. Progressive cortical lens opacification was generally observed, with the earliest onset in C57BBL/6J, C3HeB/FeJ, and 129/SvJ, between 2 and 6 months after birth. Moreover, lenses of these inbred lines developed nuclear opacities. Calculated mean lens density significantly increased between 6 and 12 months of age in all inbred strains except 129/SvJ. Lens densities (and the corresponding standard deviations) of FVB/NCrl and 129/SvJ increased most likely because of differences in the genetic background. Albinism as confounder might be excluded since the albino Balb/cByJ mice are more similar to the C57BL/6J or C3Heb/FeJ mice. We further identified strain-specific anterior lens opacities (C57BL/6J) and cloudy corneal lesions (C57BL/6J, FVB/NCrl, and BALB/cByJ) at later stages. In conclusion, our results indicate that there are lifelong opacification processes in the mouse lens. The highest lens transparency and a dark coat color, which prevents interference from light reflections, make mice with the C57BL/6J background most suitable for cataract research by Scheimpflug imaging. We show that lens densitometry by Scheimpflug imaging in mouse eyes can resolve differences of less than 1 %, making it possible to detect differences in cataract development in different mouse strains, even if they are small.     

NaCl taste thresholds in 13 inbred mouse strains

Molecular mechanisms of salty taste in mammals are not completely understood. We use genetic approaches to study these mechanisms. Previously, we developed a high-throughput procedure to measure NaCl taste thresholds, which involves conditioning mice to avoid LiCl and then examining avoidance of NaCl solutions presented in 48-h 2-bottle preference tests. Using this procedure, we measured NaCl taste thresholds of mice from 13 genealogically divergent inbred stains: 129P3/J, A/J, BALB/cByJ, C3H/HeJ, C57BL/6ByJ, C57BL/6J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/BlNJ, PWK/PhJ, and SJL/J. We found substantial strain variation in NaCl taste thresholds: mice from the A/J and 129P3/J strains had high thresholds (were less sensitive), whereas mice from the BALB/cByJ, C57BL/6J, C57BL/6ByJ, CE/J, DBA/2J, NZB/BINJ, and SJL/J had low thresholds (were more sensitive). NaCl taste thresholds measured in this study did not significantly correlate with NaCl preferences or amiloride sensitivity of chorda tympani nerve responses to NaCl determined in the same strains in other studies. To examine whether strain differences in NaCl taste thresholds could have been affected by variation in learning ability or sensitivity to toxic effects of LiCl, we used the same method to measure citric acid taste thresholds in 4 inbred strains with large differences in NaCl taste thresholds but similar acid sensitivity in preference tests (129P3/J, A/J, C57BL/6J, and DBA/2J). Citric acid taste thresholds were similar in these 4 strains. This suggests that our technique measures taste quality-specific thresholds that are likely to represent differences in peripheral taste responsiveness. The strain differences in NaCl taste sensitivity found in this study provide a basis for genetic analysis of this phenotype.     

Gene expression in lung and basal forebrain during influenza infection in mice

Inbred mice develop strain-dependent changes in sleep during the first few days after inoculation with influenza virus. To identify genes with the potential to differentially modulate sleep under this condition, we performed complementary DNA microarray analysis of both lung and basal forebrain (BF) of infected (I) and uninfected BALB/cByJ (C) and C57BL/6J (B6) mice. This analysis showed significant variation in the expression pattern of 667 and 1217 of the surveyed genes in BF and lung, respectively ( P  < 0.01). Applying the additional criterion of an effect size ≥2, 495 genes differed in expression in lung compared with 204 in BF. In BF, more genes were differentially expressed as a function of mouse strain, whereas in lung, more genes were differentially expressed as a function of health status. Significant alterations in expression after infection were more numerous and robust in BALB/cByJ vs. C57BL/6J mice. Some genes showed significant variation in both tissues as a function of strain or condition, but the changes in general were not parallel. Genes that showed significant and robust variation as a function of strain, health status or tissue included those related to immune function, metabolism, signal transduction, cell cycle regulation, apoptosis and other miscellaneous categories. Different patterns of gene expression in BF of uninfected mice suggest the possibility of fundamental mechanistic differences in pathways that modulate vigilance in these strains, whereas differences in expression of lung of infected mice suggests different peripherally generated sleep-modulatory stimuli in the two strains.     

Genetic differences in leverpress escape/avoidance conditioning in seven mouse strains

Studies of inbred mouse strains can provide us with important information about the genetic basis of learning and memory. The present experiment studies the acquisition of a leverpress escape/avoidance task in six commonly studied inbred mouse strains (C57BL/6NCrlBR, DBA/2NCrlBR, C3H/HeJ, FVB/NJ, BALB/cByJ and 129S6/SvEvTac), and one outbred strain, the CD1. Results indicated that the strains formed three discrete performance clusters. The C57BL/6NCrlBR, C3HeB/FeJ, BALB/cByJ, and CD1 strains acquired the avoidance response comparably to Sprague-Dawley rats, avoiding approximately 40% of shocks by the fourth and final training session. The 129S6/SvEvTac and FVB/NJ were extremely poor at the avoidance task throughout training. The FVB/NJ strain remained in an escape mode, while the 129S6/SvEvTac animals performed few responses of any type. Finally, the DBA/2NCrlBR strain performed exceptionally well, avoiding over 90% of the shocks by the final session. Results are discussed in terms of genetic differences in learning and how the nigrostriatal dopamine system may mediate the observed differences.     

Markers for heightened monitoring, imminent death, and euthanasia in aged inbred mice

The goal of this study was to identify objective criteria that would reliably predict spontaneous death in aged inbred mice. We evaluated male and female AKR/J mice, which die at a relatively young age due to the development of lymphoma, as well as male C57BL/6J and BALB/cByJ mice. Mice were implanted subcutaneously with an identification chip that also allowed remote measurement of body temperature. Temperatures and body weights were measured weekly until spontaneous death occurred or until euthanasia was performed for humane reasons. In AKR/J mice, hypothermia and weight loss began about 4 wk prior to death and increased gradually during that antemortem interval. In C57BL/6J and BALB/cByJ mice, these declines began earlier and were more prolonged prior to death. However, C57BL/6J and BALB/cByJ mice developed a relatively precipitous hypothermia during the 2 wk prior to death. For all 3 strains, the derived composite score of temperature × weight, expressed as a percentage of stable values for each mouse, was similarly informative. These changes in individual and composite measures can signal the need for closer observation or euthanasia of individual mice. Validated markers of clinical decline or imminent death can allow the use of endpoints that reduce terminal distress, do not significantly affect longevity or survival data, and permit timely collection of biologic samples.     

Strain-dependent airway hyperresponsiveness and a chromosome 7 locus of elevated lymphocyte numbers in cystic fibrosis transmembrane conductance regulator-deficient mice

We previously observed the lungs of naive BALB/cJ Cftr(tm1UNC) mice to have greater numbers of lymphocytes, by immunohistochemical staining, than did BALB wild type littermates or C57BL/6J Cftr(tm1UNC) mice. In the present study, we initially investigated whether this mutation in Cftr alters the adaptive immunity phenotype by measuring the lymphocyte populations in the lungs and spleens by FACS and by evaluating CD3-stimulated cytokine secretion, proliferation, and apoptosis responses. Next, we assessed a potential influence of this lymphocyte phenotype on lung function through airway resistance measures. Finally, we mapped the phenotype of pulmonary lymphocyte counts in BALB × C57BL/6J F2 Cftr(tm1UNC) mice and reviewed positional candidate genes. By FACS analysis, both the lungs and spleens of BALB Cftr(tm1UNC) mice had more CD3(+) (both CD4(+) and CD8(+)) cells than did littermates or C57BL/6J Cftr(tm1UNC) mice. Cftr(tm1UNC) and littermate mice of either strain did not differ in anti-CD3-stimulated apoptosis or proliferation levels. Lymphocytes from BALB Cftr(tm1UNC) mice produced more IL-4 and IL-5 and reduced levels of IFN-γ than did littermates, whereas lymphocytes from C57BL/6J Cftr(tm1UNC) mice demonstrated increased Il-17 secretion. BALB Cftr(tm1UNC) mice presented an enhanced airway hyperresponsiveness to methacholine challenge compared with littermates and C57BL/6J Cftr(tm1UNC) mice. A chromosome 7 locus was identified to be linked to lymphocyte numbers, and genetic evaluation of the interval suggests Itgal and Il4ra as candidate genes for this trait. We conclude that the pulmonary phenotype of BALB Cftr(tm1UNC) mice includes airway hyperresponsiveness and increased lymphocyte numbers, with the latter trait being influenced by a chromosome 7 locus.     

Characterization of single nucleotide polymorphisms for a forward genetics approach using genetic crosses in C57BL/6 and BALB/c substrains of mice

Forward genetics is a powerful approach based on chromosomal mapping of phenotypes and has successfully led to the discovery of many mouse mutations in genes responsible for various phenotypes. Although crossing between genetically remote strains can produce F₂ and backcross mice for chromosomal mapping, the phenotypes are often affected by background effects from the partner strains in genetic crosses. Genetic crosses between substrains might be useful in genetic mapping to avoid genetic background effects. In this study, we investigated single nucleotide polymorphisms (SNPs) available for genetic mapping using substrains of C57BL/6 and BALB/c mice. In C57BL/6 mice, 114 SNP markers were developed and assigned to locations on all chromosomes for full utilization for genetic mapping using genetic crosses between the C57BL/6J and C57BL/6N substrains. Moreover, genetic differences were identified in the 114 SNP markers among the seven C57BL/6 substrains from five production breeders. In addition, 106 SNPs were detected on all chromosomes of BALB/cAJcl and BALB/cByJJcl substrains. These SNPs could be used for genotyping in BALB/cJ, BALB/cAJcl, BALB/cAnNCrlCrlj, and BALB/cCrSlc mice, and they are particularly useful for genetic mapping using crosses between BALB/cByJJcl and other BALB/c substrains. The SNPs characterized in this study can be utilized for genetic mapping to identify the causative mutations of the phenotypes induced by N-ethyl-N-nitrosourea mutagenesis and the SNPs responsible for phenotypic differences between the substrains of C57BL/6 and BALB/c mice.     

Performance of BALB/c and C57BL/6 mice under an incremental repeated acquisition of behavioral chains procedure

BALB/c (n = 8) and C57BL/6 (n = 11) male mice were trained under an incremental repeated acquisition (IRA) procedure using two distinct training procedures: forward and backward chaining. A new metric for assessing progress on the IRA procedure, progress quotient (PQ), quantified progress as the product of chain length and number of reinforcers earned during a session divided by the total number of reinforcers earned. BALB/c mice progressed further, had higher overall responding, earned more reinforcers, and acquired the response sequences faster than the C57BL/6 mice on both training procedures. There were only minimal effects of training procedure for either strain. The strain differences found between BALB/c and C57BL/6 mice confirm the importance of genetic background to behavior. C57BL/6 mice may be deficient in learning as compared with BALB/c mice but other contributing factors probably include overall responding, motivation, and more rapid satiation or habituation to sucrose reinforcement by the C57BL/6 mice. PQ is a sensitive and valid measure of progress for use in studies of mastery-based incremental repeated acquisition and BALB/c mice perform this challenging learning task better than do C57BL/6 mice.     

Genetic determinants of resistance to ectromelia (mousepox) virus-induced mortality.

Resistance to ectromelia (mousepox) virus-induced mortality was examined in crosses between susceptible DBA/2J, A/J, and BALB/cByJ mice and resistant C57BL/6J and AKR/J mice. Depending on the cross, resistance to mousepox virus was shown to be determined by one or more independently assorting autosomal loci with dominant alleles for resistance in AKR/J and C57BL/6J mice and recessive alleles in A/J, BALB/cByJ, and DBA/2J mice. A sexual dimorphism in resistance to disease was also observed.     

C1q deficiency and autoimmunity: the effects of genetic background on disease expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.     

Francisella tularensis Vaccines Elicit Concurrent Protective T- and B-Cell Immune Responses in BALB/cByJ Mice

In the last decade several new vaccines against Francisella tularensis, which causes tularemia, have been characterized in animal models. Whereas many of these vaccine candidates showed promise, it remains critical to bridge the preclinical studies to human subjects, ideally by taking advantage of correlates of protection. By combining in vitro intramacrophage LVS replication with gene expression data through multivariate analysis, we previously identified and quantified correlative T cell immune responses that discriminate vaccines of different efficacy. Further, using C57BL/6J mice, we demonstrated that the relative levels of gene expression vary according to vaccination route and between cell types from different organs. Here, we extended our studies to the analysis of T cell functions of BALB/cByJ mice to evaluate whether our approach to identify correlates of protection also applies to a Th2 dominant mouse strain. BALB/cByJ mice had higher survival rates than C57BL/6J mice when they were immunized with suboptimal vaccines and challenged. However, splenocytes derived from differentially vaccinated BALB/cByJ mice controlled LVS intramacrophage replication in vitro in a pattern that reflected the hierarchy of protection observed in C57BL/6J mice. In addition, gene expression of selected potential correlates revealed similar patterns in splenocytes of BALB/cByJ and C57BL/6J mice. The different survival patterns were related to B cell functions, not necessarily to specific antibody production, which played an important protective role in BALB/cByJ mice when vaccinated with suboptimal vaccines. Our studies therefore demonstrate the range of mechanisms that operate in the most common mouse strains used for characterization of vaccines against F. tularensis, and illustrate the complexity necessary to define a comprehensive set of correlates.     

Genetic background determines metabolic phenotypes in the mouse

To evaluate the contribution of genetic background to phenotypic variation, we compared a large range of biochemical and metabolic parameters at different ages of four inbred mice strains, C57BL/6J, 129SvPas, C3HeB/FeJ, and Balb/cByJ. Our results demonstrate that important metabolic, hematologic, and biochemical differences exist between these different inbred strains. Most of these differences are gender independent and are maintained or accentuated throughout life. It is therefore imperative that the genetic background is carefully defined in phenotypic studies. Our results also argue that certain backgrounds are more suited to study a given physiologic phenomenon, as distinct mouse strains have a different propensity to develop particular biochemical, hematologic, and metabolic abnormalities. These genetic differences can furthermore be exploited to identify new genes/proteins that contribute to phenotypic abnormalities. The choice of the genetic background in which to generate and analyze genetically engineered mutant mice is important as it is, together with environmental factors, one of the most important contributors to the variability of phenotypic results.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Modification of survival rate of mouse embryos developing in heterozygous females for ovum mutant gene

The DDK syndrome (polar infertility) is caused by an incompatibility system due to the ovum mutant (Om) locus. For brevity, the following gene symbols are used in the present report: DDK allele, Om; C57BL/6Cr allele, +. In this investigation, we first attempted to introduce the Om allele of DDK strain into the genetic background of C57BL/6Cr strain. The attempt resulted in the production of no young at the third generation of successive backcrosses. Secondly, mating experiments were performed with heterozygous (Om/+) females having background genes of C57BL/6Cr and DDK strains in the ratios 1:1(B1D), 3:1(B3D), 7:1(B7D), and 15:1(B15D). The survival rate of the embryos as judged by the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy was 41.3 +/- 3.2%, 27.3 +/- 3. 2%, 16.4 +/- 3.3%, and 11.3 +/- 3.2% (mean +/- SEM) in the B1D, B3D, B7D, and B15D females, respectively, when they were mated with C57BL/6Cr males. Furthermore, the increased embryonic mortality in the heterozygous (Om/+) females with more background genes of C57BL/6Cr strain was found to be due to a failure in blastocyst formation, as in the DDK syndrome. The parallelism between the proportion of C57BL/6Cr background genes and embryonic mortality has led to a hypothesis proposing the participation of a modifier gene, namely that a mechanism similar to allelic exclusion may be working in the synthesis of cytoplasmic factor of eggs and that only the Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in heterozygous (Om/+) females having a modifier gene in the homozygous state.     

Estrous cycle effects on behavior of C57BL/6J and BALB/cByJ female mice: implications for phenotyping strategies

Systematic behavioral phenotyping of genetically modified mice is a powerful method with which to identify the molecular factors implicated in control of animal behavior, with potential relevance for research into neuropsychiatric disorders. A number of such disorders display sex differences, yet the use of female mice in phenotyping strategies has been a rare practice because of the potential variability related to the estrous cycle. We have now investigated the behavioral effects of the estrous cycle in a battery of behavioral tests in C57BL/6J and BALB/cByJ inbred strains of mice. Whereas the performance of BALB/cByJ female mice varied significantly depending on the phase of the estrous cycle in the open field, tail flick and tail suspension tests, the behavior of C57BL/6J females, with the exception of the tail suspension performance, remained stable across all four phases of the estrous cycle in all of the tests including open field, rotarod, startle reflex and pre-pulse inhibition, tail flick and hot plate. We also found that irrespective of the estrous cycle, the behavior of C57BL/6J females was different from that of BALB/cByJ groups in all of the behavioral paradigms. Such strain differences were previously reported in male comparisons, suggesting that the same inter-group differences can be revealed by studying female or male mice. In addition, strain differences were evident even for behaviors that were susceptible to estrous cycle modulations, although their detection might necessitate the constitution of large experimental groups.     

Macronutrient diet selection in thirteen mouse strains

The strain distribution for macronutrient diet selection was described in 13 mouse strains (AKR/J, NZB/B1NJ, C57BL/6J, C57BL/6ByJ, DBA/2J, SPRET/Ei, CD-1, SJL/J, SWR/J, 129/J, BALB/cByJ, CAST/Ei, and A/J) with the use of a self-selection protocol in which separate carbohydrate, fat, and protein diets were simultaneously available for 26-30 days. Relative to carbohydrate, nine strains consumed significantly more calories from the fat diet; two strains consumed more calories from carbohydrate than from fat (BALB/cByJ, CAST/Ei). Diet selection by SWR/J mice was variable over time, resulting in a lack of preference. One strain (A/J) failed to adapt to the diet paradigm due to inadequate protein intake. Comparisons of proportional fat intake across strains revealed that fat selection/consumption ranged from 26 to 83% of total energy. AKR/J, NZB/B1NJ, and C67BL/6J mice self-selected the highest proportion of dietary fat, whereas the CAST/Ei and BALB/cByJ strains chose the lowest. Finally, epididymal fat depot weight was correlated with fat consumption. There were significant positive correlations in AKR/J and C57BL/6J mice, which are highly sensitive to dietary obesity. However, absolute fat intake was inversely correlated with epididymal fat in two of the lean strains: SWR/J and CAST/Ei. We hypothesize that the SWR/J and CAST/Ei strains are highly sensitive to a negative feedback signal generated by increasing body fat, but the AKR/J and C67BL/6J mice are not. The variation in dietary fat selection across inbred strains provides a tool for dissecting the complex genetics of this trait.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.