C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund's Adjuvant model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic constrictive nerve injury, a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-chronic constrictive nerve injury. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J × B6N-F2 cross (N = 164), we mapped a major quantitative trait locus underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (log of the odds [LOD] = 3.81, p < 0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression quantitative trait loci associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (false discovery rate < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

What's in a (Sub)strain?

C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of Stem Cell Reports, Morales-Hernández et al. identify one particular difference-disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)-that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice.

Effects of Repeated Anesthesia Containing Urethane on Tumor Formation and Health Scores in Male C57BL/6J Mice.

Repeated injection of urethane (ethyl carbamate) is carcinogenic in susceptible strains of mice. Most recent cancer studies involving urethane-induced tumor formation use p53(+/-) mice, which lack one copy of the p53 tumor suppressor gene. In contrast, the same protocol elicits at most a single tumor in wildtype C57BL/6 mice. The effect of repeatedly injecting urethane as a component of a ketamine-xylazine anesthetic mixture in the highly prevalent mouse strain C57BL/6 is unknown. Male C57BL/6J mice (n = 30; age, 3 mo) were anesthetized once monthly for 4 mo by using 560 mg/kg urethane, 28 mg/kg ketamine, and 5.6 mg/kg xylazine. The physical health of the mice was evaluated according to 2 published scoring systems. The average body condition score (scale, 1 to 5; normal, 3) was 3.3, 3.3, and 3.4 after the 2nd, 3rd, and 4th injections, respectively. The visual assessment score was 0 (that is, normal) at all time points examined. Within 1 wk after the 4th injection, the mice were euthanized, necropsied, and evaluated histopathologically. No histopathologic findings were noteworthy. We conclude that repeated monthly injection with urethane as a component of an anesthetic cocktail does not cause clinically detectable abnormalities or induce neoplasia in C57BL/6J mice. These findings are important because urethane combined with low-dose ketamine, unlike other anesthetic regimens, allows for accurate recording of neuronal activity in both the brain and retina. Longitudinal neuronal recordings minimize the number of mice needed and improve the analysis of disease progression and potential therapeutic interventions.

Neocortical molecular layer heterotopia in substrains of C57BL/6 and C57BL/10 mice.

Abnormal development of the neocortex is often associated with cognitive deficits and epilepsy. Rodent models are widely used to study normal and abnormal cortical development and have revealed the roles of many important genetic and environmental factors. Interestingly, several inbred mouse strains commonly used in behavioral, anatomical, and/or physiological studies display neocortical malformations including C57BL/6J mice, which are among the most widely utilized mice. In the present report we describe the prevalence and cytoarchitecture of molecular-layer heterotopia in C57BL/6J mice and related strains obtained from three commercial vendors as well as mice bred in academic vivaria from founders obtained commercially. In particular, we found that the prevalence of molecular-layer heterotopia vaired according to the sex as well as the vendor-of-origin of the mouse. These data are relevant to the use of this strain as a mouse-model in the study of brain-behavior relationships.

The blessings and curses of C57BL/6 substrains in mouse genetic studies.

Phenotypic and genetic differences among C57BL/6 substrains are accumulating. Investigators must address these differences to improve the quality of their studies.

Agouti C57BL/6N embryonic stem cells for mouse genetic resources.

We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.

Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.

Traditional protocols for sperm recovery, cryopreservation, and in vitro fertilization (IVF) have been considerably less efficient for inbred mouse strains, including C57BL/6, than for hybrid and outbred strains. We report here that 3 changes to published and widely used protocols markedly improved fertilization rates for both fresh and frozen-thawed sperm in 3 substrains of C57BL/6 mice (C57BL/6J, C57BL/6NCrl, and C57BL/6NTac). First, the traditional cyroprotective agent was modified by adding amino acids. Second, preincubation of sperm in a preincubation medium containing methyl-beta-cyclodextrin and polyvinyl alcohol enabled collection of progressively motile sperm for IVF. Third, we evaluated 3 media for IVF: human tubal fluid (HTF), modified Krebs-Ringer bicarbonate medium (TYH), and minimal essential medium (MEM). HTF and TYH were modified by adding minimal essential amino acids. The methodology reported here increased the IVF rate of both fresh and frozen-thawed sperm and enabled efficient isolation of capacitated viable sperm. Fertilization rates greater than 65% and 40% were obtained with the 3 tested substrains when fresh and frozen-thawed sperm, respectively, were used for IVF. Higher fertilization rates were seen with frozen-thawed sperm from C57BL/6NCrl and C57BL/6NTac mice than from C57BL/6J mice. Among all strains, fresh sperm from C57BL/6NTac mice gave the highest fertilization rate. Of 190 two-cell embryos, 63 (33.2%) developed to term after transfer to pseudopregnant recipient mice. The protocol we detail here provides reliable cryopreservation and recovery of live mice in 3 substrains of C57BL/6, making sperm cryopreservation and IVF a viable choice for preservation and distribution of mouse lines.

Recurrent DNA copy number variation in the laboratory mouse.

Different species, populations and individuals vary considerably in the copy number of discrete segments of their genomes. The manner and frequency with which these genetic differences arise over generational time is not well understood. Taking advantage of divergence among lineages sharing a recent common ancestry, we have conducted a genome-wide analysis of spontaneous copy number variation (CNV) in the laboratory mouse. We used high-resolution microarrays to identify 38 CNVs among 14 colonies of the C57BL/6 strain spanning approximately 967 generations of inbreeding, and we examined these loci in 12 additional strains. It is clear from our results that many CNVs arise through a highly nonrandom process: 18 of 38 were the product of recurrent mutation, and rates of change varied roughly four orders of magnitude across different loci. Recurrent CNVs are found throughout the genome, affect 43 genes and fluctuate in copy number over mere hundreds of generations, observations that raise questions about their contribution to natural variation.

Cytochemical analysis of pancreatic islet hypercytolipidemia following diabetes (db/db) and obese (ob/ob) mutation expression: influence of genomic background.

Both diabetes (db/db) and obese (ob/ob) genotype mutations induce a hyperglycemic-hyperinsulinemic endometabolic state in C57BL mice, manifesting a type II NIDDM diabetes-obesity syndrome (DOS) in these leptin ligand/receptor-deficient models. The severity of the DOS induced by these single gene, homozygous-recessive mutations may be moderated by the background genome on which the mutation is expressed. The current studies define the phenotypic, systemic, cytochemical and cellular metabolic responses to db/db and ob/ob mutation expression when modified by /KsJ (severe DOS expression) or /6 (modified DOS expression) background strain influences as compared to littermate control (+/?) indices. Both db/db and ob/ob mutations induced dramatic increases in body weights, blood glucose and serum insulin concentrations relative to +/? indices when expressed on either the C57BL/KsJ (-/KsJ) or C57BL/6 (-/6) backgrounds. However, the -/KsJ background enhanced the severity of expression of these DOS indices relative to the -/6 strain. Similarly, the -/KsJ genome suppressed cellular glucose uptake rates, pancreatic tissue weights and insulin concentrations in both db/db and ob/ob mutants relative to /6 background strain influences or +/? indices. Concurrent enhancement of tissue and cellular lipogenic metabolism and islet cytolipid depositions were exaggerated when the mutations were expressed on the -/KsJ background relative to the -/6 genome. Pancreatic islet B-cell lipodeposition was markedly enhanced in ob/ob and db/db mutants expressed on either the -/KsJ or -/6 background. In both ob/ob and db/db models, B-cell insulin granulation was prominent in mildly hypertrophic pancreatic islets when the mutations were expressed on the -/6 background. In contrast, the severity of the DOS state expressed on the -/KsJ background resulted in pronounced B-cell atrophy, characterized by insulin degranulation, cellular hypertrophy and hypercytolipidemia associated with tissue involution, in both ob/ob and db/db mutants. Dramatic alterations in tissue norephinephrine (NE) and alpha-1-receptor populations in ob/ob and db/db mutants were exaggerated by the -/KsJ genome as compared to -/6 or control indices. The influences of the -/KsJ genome on the progressive expression of tissue NE counter-regulatory responses to enhanced cytolipidemic indices were inversely related, with cytochemical lipodeposition occurring under conditions of diminished adrenergic responses to the DOS indices. The results of these studies indicate that the severity of the type-II diabetes endometabolic syndrome induced by the ob/ob or db/db genotypic mutations is modified by the existing genome on which the mutations are expressed. These data suggest that the severity of genomic mutation expression may be modified depending on the capability of the background genome to counter-regulate the systemic, cellular or metabolic consequences of these mutations.

Signal sequence conservation and mature peptide divergence within subgroups of the murine beta-defensin gene family.

Beta-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six beta-defensin exon 2-like sequences and subsequently found full-length expressed transcripts for these novel genes. Expression was high in brain and reproductive tissues. Eleven beta-defensins could be grouped into two clear subgroups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, however, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Examination of the gene sequences of orthologs in other rodents also revealed an excess of nucleotide changes that altered amino acids in the mature peptide region. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding noncoding DNA show little divergence within subgroups. The focus for rapid sequence divergence is localized in the DNA encoding the mature peptide and this is driven by accelerated positive selection. This mechanism of evolution is consistent with the role of this gene family as defense against bacterial pathogens and the sequence changes have implications for novel antibiotic design.

Ethanol-induced expression of c-Fos differentiates the FAST and SLOW selected lines of mice.

The effect of ethanol on the number of Fos-like immunoreactive (Fos-li) neurons was previously studied in the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains (Hitzemann and Hitzemann, 1997). Data obtained suggested that the locomotor activation response to ethanol found in the D2 but not the B6 strain was associated with an increase in the number of Fos-li neurons (a putative measure of synaptic activity) in the central nucleus of the amygdala (CeA), but not in other brain regions, including the basal ganglia. Supporting results were obtained in B6D2 F2 intercross animals (Demarest et al., 1998) those animals exhibiting a marked locomotor activation response to ethanol also showed a significant increase in the number of Fos-li neurons in the CeA. The current study extends this line of investigation to the FAST and SLOW selected lines of mice (Shen et al., 1995). Twenty-eight SLOW and FAST mice (taken evenly from both replicate lines) were randomly assigned to receive either saline or ethanol (1.5 g/kg). One hour later, the animals were sacrificed, and the number of Fos-li neurons were determined using standard immunocytochemical techniques. Both the FAST and SLOW lines showed a marked increase (>300%) in the number of Fos-li neurons in the lateral aspect of the CeA; however, in the capsular division, only the FAST line showed an increase (>500%). In several brain regions, the basal (saline) response was markedly higher in the SLOW line; these regions included the subthalamic nucleus, the entopeduncular nucleus, the substantia nigra compacta, and the ventral tegmental area. Furthermore, it was found that ethanol decreased the number of Fos-li neurons in the ventral tegmental area of the SLOW but not FAST mice. These data suggest a substantial involvement of the basal ganglia in the segregation of the FAST and SLOW lines.

Immune response to murine neuroblastoma: effects of in vitro culture of the tumor. Mitogen and mixed lymphocyte culture responses, interleukin-2 production, suppression and natural killer activity.

The cellular immune response in A/J mice inoculated with in vitro cultured neuroblastoma (NB) cells is completely different from the response obtained after inoculation of in vivo passaged NB cells. Indeed, inoculation of in vivo grown tumor cells leads to a strongly decreased MLC and PHA response of the host spleen cells. This depressed response is not reversed by in vitro addition of indomethacin, but correlates with a low IL-2 production by the spleen cells. In addition, non-specific suppressor cells are present in the spleen and in the peritoneal cavity. The splenic suppressor cells are of low specific gravity and they are nylon wool adherent. The suppressor capacity is not reduced by anti-Thy plus rabbit complement treatment. Another effect of in vivo maintained NB cells is that the natural killer (NK) activity of the spleen cells of the host is increased 4-5 fold 2 days after inoculation of the tumor cells. On the contrary, in vitro passaged NB cells do not induce a reduced MLC and PHA responsiveness, and the IL-2 production of the host spleen cells remains nearly normal. No non-specific suppression can be demonstrated and the NK activity is not augmented 2 days after inoculation. We can conclude that it is very important in which condition tumor cells are maintained when used to study anti-tumor immunity.

Serological biochemical and functional characterisation of three different HLA-DR monoclonal antibodies derived from C57BL6 mice.

C57BL6 mice which do not express I-E gene products were immunised with EBV transformed human B cell lines to generate MoAbs. Three hybridoma supernatants which initially reacted with the immunising donor cell but not a T cell line lacking Class II antigens were further investigated. I-D SDS-PAGE patterns of molecules precipitated by the three supernatants from a cell membrane lysate were characteristic of HLA-Class II alpha and beta chains. Two-dimensional analysis established the specificity of the supernatants as HLA-DR specific. This was confirmed by the reaction patterns with Class II mutant deletant cell lines. In both ELISA and cytotoxicity one reacted with all lymphoblastoid cell lines tested, one reacted with all except two that were DR7 homozygous and the third reacted strongly only with cells that were DR3. All three antibodies were cytotoxic to both peripheral blood lymphocytes and EBV transformed B cell lines. The DR3 specific MoAb (IgG2a) was suitable as a typing reagent. The DR3 reactive MoAb specifically inhibited stimulation by a Dw3 HTC and the other two MoAbs inhibited all HTCs tested. These findings are consistent with the view that certain determinants responsible for the Dw specificities are carried on the DR molecules.

Local interferon induction by bacterial lipopolysaccharide in mice after pretreatment with Corynebacterium parvum.

C57BL/6 mice were injected intraperitoneally (i.p.) with Corynebacterium parvum and subsequently, after an interval of 7-10 days, i.p. with lipopolysaccharide (LPS). The peritoneal wash-fluid was recovered at various times after injection of LPS. Marked interferon (IFN) titers were observed between 2 and 10 h after injection of LPS, whereas no IFN was detected in mice injected with either C. parvum or LPS alone. Very low doses of LPS (0.1 microgram/mouse) were sufficient to cause IFN production in the double-stimulation protocol. The IFN produced was neutralized by an antibody against IFN-alpha/beta. In additional experiments, mice were treated by C. parvum alone; the peritoneal exudate cells (PEC) were recovered and stimulated in vitro by LPS. Again substantial titers of IFN were induced by small concentrations of LPS, whereas untreated PEC did not produce IFN. The cell producing IFN in these cultures was not a T lymphocyte, as experiments with a monoclonal anti-thy 1.2 antibody showed.

Parallelism between superoxide production of peritoneal exudate cells and lung granulomatous response in mice vaccinated with BCG cell walls.

Since peritoneal macrophages are reported to be different from alveolar macrophages in their activated states, we examined whether O-2 production, one of the parameters of macrophage activation, in mouse peritoneal exudate cells (PEC) is enhanced under the condition in which lung granuloma, the accumulation of activated macrophages, is produced with Bacillus Calmette-Guérin (BCG) cell wall (CW). As a result, we observed the enhanced O-2 production of PEC that occurs in parallel with lung granuloma formation; high responders, C56BL/6 mice, showed high O-2 production of PEC whereas low responders, C3H/He and DBA/1 mice showed low O-2 production of PEC, suggesting that enhanced O-2 production of PEC as well as lung granuloma formation is genetically controlled. Results from T cell-depleted mice and allogeneic bone marrow chimeric mice also showed the occurrence of this parallelism. From these findings, we presumed that circulating macrophage activating factor and other lymphokines produced by BCG CW-sensitized T cells may activate both peritoneal macrophages and lung macrophages.

Functional comparison of bone marrow-derived macrophages obtained by cultivation in serum-free or serum-supplemented medium.

Bone marrow-derived macrophages obtained by cultivation in a serum-free or in a serum-supplemented medium were compared in terms of the activation of the respiratory burst and the activation of tumor cytotoxicity. Serum-free-cultured macrophages responded to interferon-gamma (IFN-gamma) and to lipopolysaccharide (LPS) by an enhancement of the respiratory burst. Macrophages obtained in a serum-supplemented medium are characterized by a diminished capacity to release O2-. These cells did not respond to IFN-gamma unless the stimulation was performed in a serum-containing medium. In terms of activation of tumor cell cytotoxicity, serum-supplemented macrophage cultures seem to be primed by unknown serum constituents because they only need one signal (IFN-gamma or LPS) to become fully cytotoxic. Serum-free cultivated macrophages can be rendered cytotoxic only after exposure to combinations of IFN-gamma and LPS.

Influence of surgical stress on cellular immunity and the induction of plastic adherent suppressor cells of spleen in mice.

Surgical stress animal models were established by performing laparotomies on mice. It was found that this type of stress could suppress the natural killer (NK) cytotoxicity and the proliferation of spleen cells induced by conA or lipopolysaccharide (LPS). Dynamic changes showed that the stress-mediated immunosuppression was reversible, as the responses to conA and LPS would be restored with time. The sensitivity to the stress-mediated suppression was different according to variations in immunological parameters. Furthermore, the macrophages in spleen were tested by isolation by the plastic-adherent procedure. The results showed clearly that these adherent cells (Plastic Adherent Cells, PAC) possessed an immunosuppressive effect on mitogen-induced lymphocyte proliferation in post-operative mice, but not in normal mice. Treatment of mice with indomethacin blocked the PAC-mediated immunosuppression. Surgical stress appeared to increase the level of prostaglandins, which in turn induced the production of suppressor PAC.

Effects of cyclosporin A on the activity of mouse natural killer cells and hybrid resistance.

It was found that a single dose of cyclosporin A (CSA) administered intraperitoneally resulted in rapid, but transitory reduction of (C57B1/6 X DBA/2)F1 spleen cell natural killer (NK) activity (on day 1 after CSA), then a return to the normal level (on day 3 after CSA) and finally gradual, but sharp decrease of this activity (the lowest activity on day 9). It was also found that CSA injected 3 days before semi-allogenic bone marrow transplantation (BMT) has no effect on hybrid resistance (HR), but administered 9 days before BMT caused abrogation of HR. So, there was a correlation between changes of NK activity after CSA and effects of CSA on HR, suggesting that NK cells are responsible for HR. In contrast, there was no effect of CSA on the engraftment of syngeneic bone marrow cells.