Common Laboratory Mouse Strains Research Articles

The C886T Mutation in the Th Gene Reduces the Activity of Tyrosine Hydroxylase in the Mouse Brain

Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse Th gene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST) mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA levels and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity. Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been demonstrated. These results will help to understand the role of TH in the development of adaptive and pathological behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents for modulating its function.

Active Induction of a Multiple Sclerosis-Like Disease in Common Laboratory Mouse Strains.

Experimental autoimmune encephalomyelitis (EAE) is a neuroinflammatory disease with facets in common with multiple sclerosis (MS). It is induced in susceptible mammalian species, with rodents as the preferred hosts, and has been used for decades as a model to investigate the immunopathogenesis of MS as well as for preclinical evaluation of candidate MS therapeutics. Most commonly, EAE is generated by active immunization with central nervous system (CNS) antigens, such as whole CNS homogenate, myelin proteins, or peptides derived from these proteins. However, EAE actually represents a spectrum of diseases in which specific combinations of host/CNS antigen exhibit defined clinical profiles, each associated with unique immunological and pathological features. Similar to MS, EAE is a complex disease where development and progression are also modulated by environmental factors; therefore, the establishment of any given EAE variant can be challenging and requires careful optimization. Here, we describe protocols for three EAE variants, successfully generated in our laboratory, and provide additional information as to how to maintain their unique features and reproducibility.

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes.

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Abstract 1384: Immunopeptidome analysis of mouse MHC Class I

Abstract MHC class I is required for antigen presentation to CD8 T cells. These cell-surface proteins are heterodimers consisting of a variable alpha chain associated with β2 microglobulin and are expressed on nucleated cells at varying levels. The antigen specificity of the interaction between MHC, peptide and T cell receptor can be exploited in development of personalized cancer vaccines and other immunotherapies against cancer. MHC sequences are species-specific and there are many different haplotypes of MHC class I. For this reason, the antibodies used for immunoprecipitation of MHC complexes are a critical component of the workflow for identification of antigens presented by MHC. Subclasses of mouse MHC class I are divided into classical (H-2D, H-2K and H-2L) and non-classical (H-2Q, H-2M and H-2T), and haplotypes of each are known for homozygous inbred laboratory strains of mice. One common strain is C57BL/6, which expresses H2-Db and H2-Kb classical MHC molecules. Antibody clone M1/42, which reacts with H-2 expressed on nucleated cells from mice of a, b, d, j, k, s and u haplotypes, is commonly referred to as a pan-MHC class I antibody. Antibody clones Y-3 and HB-27 recognize H2-Kb and H2-Db haplotypes, respectively. In this study we aimed to compare different antibodies to mouse MHC class I complexes for their efficiency of recovery of associated peptides. Input was murine EL-4 cells which are T lymphoblast cells derived from a C57BL/6 background. We performed immunoprecipitation with different antibodies either singly or sequentially to enrich MHC class I, followed by gentle elution of associated peptides and finally peptide analysis by mass spectrometry. Each immunoprecipitation workflow yielded a different (but overlapping) cohort of peptides, suggesting optimal methodologies for analyses of immunopeptidomes from common laboratory mouse strains. Citation Format: Seeta Nyayapathy, Richard Jones, Michael Ford, Michael Pisano, Julie M. Rumble. Immunopeptidome analysis of mouse MHC Class I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1384.

Characterization of the blood\u2013brain barrier in genetically diverse laboratory mouse strains

BackgroundGenetic variation in a population has an influence on the manifestation of monogenic as well as multifactorial disorders, with the underlying genetic contribution dependent on several interacting variants. Common laboratory mouse strains used for modelling human disease lack the genetic variability of the human population. Therefore, outcomes of rodent studies show limited relevance to human disease. The functionality of brain vasculature is an important modifier of brain diseases. Importantly, the restrictive interface between blood and brain—the blood–brain barrier (BBB) serves as a major obstacle for the drug delivery into the central nervous system (CNS). Using genetically diverse mouse strains, we aimed to investigate the phenotypic and transcriptomic variation of the healthy BBB in different inbred mouse strains.MethodsWe investigated the heterogeneity of brain vasculature in recently wild-derived mouse strains (CAST/EiJ, WSB/EiJ, PWK/PhJ) and long-inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, DBA/2J, NOD/ShiLtJ) using different phenotypic arms. We used immunohistochemistry and confocal laser microscopy followed by quantitative image analysis to determine vascular density and pericyte coverage in two brain regions—cortex and hippocampus. Using a low molecular weight fluorescence tracer, sodium fluorescein and spectrophotometry analysis, we assessed BBB permeability in young and aged mice of selected strains. For further phenotypic characterization of endothelial cells in inbred mouse strains, we performed bulk RNA sequencing of sorted endothelial cells isolated from cortex and hippocampus.ResultsCortical vessel density and pericyte coverage did not differ among the investigated strains, except in the cortex, where PWK/PhJ showed lower vessel density compared to NOD/ShiLtJ, and a higher pericyte coverage than DBA/2J. The vascular density in the hippocampus differed among analyzed strains but not the pericyte coverage. The staining patterns of endothelial arteriovenous zonation markers were similar in different strains. BBB permeability to a small fluorescent tracer, sodium fluorescein, was also similar in different strains, except in the hippocampus where the CAST/EiJ showed higher permeability than NOD/ShiLtJ. Transcriptomic analysis of endothelial cells revealed that sex of the animal was a major determinant of gene expression differences. In addition, the expression level of several genes implicated in endothelial function and BBB biology differed between wild-derived and long-inbred mouse strains. In aged mice of three investigated strains (DBA/2J, A/J, C57BL/6J) vascular density and pericyte coverage did not change—expect for DBA/2J, whereas vascular permeability to sodium fluorescein increased in all three strains.ConclusionsOur analysis shows that although there were no major differences in parenchymal vascular morphology and paracellular BBB permeability for small molecular weight tracer between investigated mouse strains or sexes, transcriptomic differences of brain endothelial cells point to variation in gene expression of the intact BBB. These baseline variances might be confounding factors in pathological conditions that may lead to a differential functional outcome dependent on the sex or genetic polymorphism.

Biological sex, not reproductive cycle, influences peripheral blood immune cell prevalence in mice.

Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex. Immunophenotyping (i.e. quantifying the number and types of circulating leukocytes) is used to characterize immune changes during health and disease, and in response to pharmacological and other interventions. Despite the importance of biological sex in immune function, there is considerable uncertainty amongst researchers as to the extent to which biological sex or the female reproductive cycle influence blood immunophenotype. We quantified circulating leukocytes by multicolour flow cytometry in young C57BL/6J mice and assessed the effects of the reproductive cycle, biological sex, and other experimental and biological factors on data variability. We found that there are no significant effects of the female reproductive cycle on the prevalence of peripheral blood B cells, NK cells, CD4+ T cells, CD8+ T cells, monocytes, or neutrophils. Immunophenotype composition and variability do not significantly change between stages of the female reproductive cycle. There are, however, sex-specific differences in immune cell prevalence, with fewer monocytes, neutrophils, and NK cells in female mice. Surprisingly, immunophenotype is more variable in male mice, and weight is a significant contributing factor. We provide tools for researchers to perform a priori sample size calculations for two-group and factorial analyses. We show that immunophenotype varies between inbred mouse strains, and that using equal sample sizes of male and female mice is not always appropriate for within-sex evaluations of immune cell populations in peripheral blood.

Mouse strain-dependent egg factors regulate calcium signals at fertilization.

Calcium (Ca2+ ) signals triggered at fertilization initiate resumption of the cell cycle and initial steps of embryonic development. In mammals, the sperm factor phospholipase Cζ triggers the release of Ca2+ from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca2+ transients that is modulated by egg factors including Ca2+ influx channels, Ca2+ transporters, and phosphoinositide-regulating enzymes. Here we compared characteristics of Ca2+ oscillations following in vitro fertilization (IVF) and ER Ca2+ stores among nine common laboratory mouse strains: CF1, C57BL6, SJL, CD1, DBA, FVB, 129X1, BALBc, 129S1, and the F1 hybrid B6129SF1. Sperm from B6SJLF1/J males was used for all IVF experiments. There were significant differences among the strains with respect to duration and maximum amplitude of the first Ca2+ transient, frequency of oscillations, and ER Ca2+ stores. With male strain held constant, the differences in Ca2+ oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg-intrinsic properties in determining Ca2+ oscillation patterns and have important implications for the interpretation and comparison of studies on Ca2+ dynamics at fertilization.

NSG mice as hosts for oncological precision medicine

Patient-derived xenograft (PDX) models have been rediscovered as meaningful research tool. By using severely immunodeficient mice, high-engraftment rates can be theoretically achieved, permitting clinical stratification strategies. Apart from engraftment efficacy, tolerability towards certain cytostatic drugs varies among individual mouse strains thus impeding large-scale screenings. Here, we aimed at optimizing an in vivo treatment schedule using the widely applied cytostatic drug 5-fluoruracil (5-FU) for exemplary response prediction in colorectal cancer (CRC) PDX models. Four different individual CRC PDX models were engrafted into NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice. Mice with established PDX were allocated to different treatment groups, receiving 5-FU, the oral prodrug Capecitabine, or 5-FU/leucovorin (LV) at different doses. Body weight, tumor size, and general behavior were assessed during therapy. Ex vivo analyses were done from blood samples, liver, as well as tumor resection specimen. Engraftment efficacy was high as expected in NSG mice, yielding stable PDX growth for therapy stratification. However, overall tolerability towards 5-FU was unexpectedly low, whereas the prodrug Capecitabine as well as the combination of 5-FU/LV at low doses were well tolerated. Accompanying plasma level determination of DYPD, the rate-limiting enzyme for 5-FU-mediated toxicity, revealed reduced activity in NSG mice compared with other common laboratory mouse strains, offering a likely explanation for the drug incompatibility. Also, the De Ritis quotient was highly elevated in treated mice, reflecting overall organ injury even at low doses. Summarizing these findings, NSG mice are ideal hosts for in vivo engraftment studies. However, the complex immunodeficiency reduces tolerance to certain drugs, thus making those mice especially sensitive. Consequently, such dose finding and tolerance tests constitute a necessity for similar cancer precision medicine approaches.

Contrasting characteristic behaviours among common laboratory mouse strains.

Mice are widely used to model wide-ranging human neurological disorders, from development to degenerative pathophysiology. Behavioural and molecular characteristics of these mouse models are influenced by the genetic background of each strain. Among the most commonly used strains, the inbred C57BL/6J, BALB/c, CBA and 129SvEv lines and the CD1 outbred line are particularly predominant. Despite their prevalence, comparative performance of these strains on many standard behavioural tests commonly used to assess neurological conditions remains diffusely and indirectly accessible in the literature. Given that independent studies may be conducted with mice of differing genetic backgrounds, any variation in characteristic behavioural responses of specific strains should be delineated in order to properly interpret results among studies. Thus, in the present study, we aimed to characterize these commonly used mice strains through several standard behavioural tests. Here, we found that animals from different genetic background strains exhibited varying behavioural patterns when assessed for sociability/novelty, memory function, and negative behaviours like despair and stress calls. These results suggest that genetic variation among strains may be responsible—in part—for strain-specific behavioural phenotypes and potential predisposition to some neurological disorders.

Profiling and genetic control of the murine immunoglobulin G glycome.

Immunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter-individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences; (ii) five genetic loci were found to be associated with murine IgG glycosylation; (iii) variants outside traditional glycosylation site motifs affected glycome variation; (iv) bisecting N-acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.

Deep genome sequencing and variation analysis of 13 inbred mouse strains defines candidate phenotypic alleles, private variation and homozygous truncating mutations.

BackgroundThe Mouse Genomes Project is an ongoing collaborative effort to sequence the genomes of the common laboratory mouse strains. In 2011, the initial analysis of sequence variation across 17 strains found 56.7 M unique single nucleotide polymorphisms (SNPs) and 8.8 M indels. We carry out deep sequencing of 13 additional inbred strains (BUB/BnJ, C57BL/10J, C57BR/cdJ, C58/J, DBA/1J, I/LnJ, KK/HiJ, MOLF/EiJ, NZB/B1NJ, NZW/LacJ, RF/J, SEA/GnJ and ST/bJ), cataloguing molecular variation within and across the strains. These strains include important models for immune response, leukaemia, age-related hearing loss and rheumatoid arthritis. We now have several examples of fully sequenced closely related strains that are divergent for several disease phenotypes.ResultsApproximately 27.4 M unique SNPs and 5 M indels are identified across these strains compared to the C57BL/6 J reference genome (GRCm38). The amount of variation found in the inbred laboratory mouse genome has increased to 71 M SNPs and 12 M indels. We investigate the genetic basis of highly penetrant cancer susceptibility in RF/J finding private novel missense mutations in DNA damage repair and highly cancer associated genes. We use two highly related strains (DBA/1J and DBA/2J) to investigate the genetic basis of collagen-induced arthritis susceptibility.ConclusionsThis paper significantly expands the catalogue of fully sequenced laboratory mouse strains and now contains several examples of highly genetically similar strains with divergent phenotypes. We show how studying private missense mutations can lead to insights into the genetic mechanism for a highly penetrant phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1024-y) contains supplementary material, which is available to authorized users.

Variability of brain anatomy for three common mouse strains

The way in which brain structures express different morphologies is not fully understood. Here we investigate variability in brain anatomy using ex vivo MRI of three common laboratory mouse strains: in two inbred strains (C57BL/6 and 129S6) and one outbred strain (CD-1). We use Generalised Procrustes Analysis (GPA) to estimate modes of anatomical variability. We find three distinct bilateral modes of anatomical surface variability associated with the motor cortex, the anterior somatosensory, the retrosplenial and the entorhinal cortex. The modes of variability that are associated with the motor cortex and anterior somatosensory cortex are predominantly due to genetic, i.e. strain differences. Next, we specifically test if a particular strain is more variable. We find that only the mode associated with motor cortex size has a slightly larger variance in the outbred CD-1 mice compared to the two inbred strains. This suggests that the hypothesis that outbred strains are more variable in general is not true for brain anatomy and the use of outbred CD-1 mice does probably not come at the price of increased variability. Further, we show that the first two principal components distinguish between the three strains with 91% accuracy. This indicates that neuroanatomical strain differences are captured by considerably fewer dimensions than necessary for atlas-based or voxel-wise testing. Statistical comparisons based on shape models could thus be a powerful complement to traditional atlas and voxel-based methods at detecting gene-related brain differences in mice. Finally, we find that the principal components of individual brain structures are correlated, suggesting a tightly coupled network of interdependent developmental trajectories. These results raise the question to what degree neuroanatomical variability is directly genetically determined or the result of experience and epigenetic mechanisms.

Abstract 12587: Telomere Length Regulates Cellular Fate of Cardiac Progenitor Cells

Myocardial regeneration following injury is severely limited by aging. Understanding the impact of age-associated factors such as telomere shortening is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs), which are currently used in clinical trials. Telomere length (TL) determines the proliferation potential such that short telomeres cause senescence of CPCs, however the impact of TL on regulating the balance between quiescence and differentiation of adult stem cells remains undetermined. We hypothesize that TL regulates the equilibrium between quiescence, proliferation, commitment and senescence, thus determining cellular fate of CPCs. CPCs were isolated from 3 mouse strains with varying TLs- common laboratory mouse strains FVB (TL:70Kb) and C57 (TL:50Kb), as well as a naturally occurring wild mouse strain, Mus musculus castaneus (CAST) possessing critically short telomeres (TL:18Kb) comparable to humans (TL:5-10Kb). CAST CPCs with short TLs have slower proliferation rate (-80% vs FVB, p<0.01), increased population doubling time and exhibit characteristics of senescence such as flattened cell morphology, increased beta-galactosidase activity (p<0.05) and p53 expression (1.7 fold, p<0.05). Interestingly, CAST CPCs also undergo basal differentiation in the absence of commitment cues, as evidenced by increased expression of lineage markers smooth muscle actin (SMA; 16.6 fold, p<0.01), Tie2 (1.7 fold, p<0.05), and sarcomeric actinin (1.75 fold). Acquisition of differentiation and senescence is accompanied by reduced expression of quiescence marker p21 (-33%). Consistent findings of senescence, increased basal differentiation (11.3 and 1.6 fold increase in SMA and Tie2 levels) and loss of quiescence (-26% decrease in p21 levels) are evidenced in CPCs isolated from old FVB mice with short TLs relative to young CPCs. Corroborative findings in adult human failing heart CPCs with short TLs suggests that telomere shortening tips the equilibrium away from quiescence and proliferation into differentiation and senescence leading to exhaustion of CPCs. Understanding mechanistically how TL regulates CPC fate will enable identification of molecular strategies to enhance the therapeutic effects of CPCs.

The Mouse Genomes Project: a repository of inbred laboratory mouse strain genomes.

The Mouse Genomes Project was initiated in 2009 with the goal of using next-generation sequencing technologies to catalogue molecular variation in the common laboratory mouse strains, and a selected set of wild-derived inbred strains. The initial sequencing and survey of sequence variation in 17 inbred strains was completed in 2011 and included comprehensive catalogue of single nucleotide polymorphisms, short insertion/deletions, larger structural variants including their fine scale architecture and landscape of transposable element variation, and genomic sites subject to post-transcriptional alteration of RNA. From this beginning, the resource has expanded significantly to include 36 fully sequenced inbred laboratory mouse strains, a refined and updated data processing pipeline, and new variation querying and data visualisation tools which are available on the project's website ( http://www.sanger.ac.uk/resources/mouse/genomes/ ). The focus of the project is now the completion of de novo assembled chromosome sequences and strain-specific gene structures for the core strains. We discuss how the assembled chromosomes will power comparative analysis, data access tools and future directions of mouse genetics.

Lrit3 Deficient Mouse (nob6): A Novel Model of Complete Congenital Stationary Night Blindness (cCSNB)

Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade.

Next-generation sequencing of experimental mouse strains

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5 years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.

Immunological Variation Between Inbred Laboratory Mouse Strains

Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.

Systematic analysis of histamine and N-methylhistamine concentrations in organs from two common laboratory mouse strains: C57Bl/6 and Balb/c

Histamine plays a role in several (patho) physiological processes that are commonly studied in mouse models. However, a systematic quantification of histamine and its metabolite N-methylhistamine in mouse organs has not been reported so far. Balb/c and C57Bl/6 mice were grouped according to their sex and age. Brains, hearts, lungs, livers, kidneys, stomachs, intestines, thymi, spleens, and lymph nodes were excised, weighed, and homogenized. Histamine and N-methylhistamine were quantified simultaneously by a HPLC-mass spectrometry method. In all organs analyzed, histamine and N-methylhistamine were detected; however, with quantitative differences. Histamine was present most abundantly in the stomach, lymph nodes, and thymus. The lowest histamine concentrations were detected in brain, liver, lung, and intestine. In most organs, the histamine concentrations increased age-dependently. Substantial concentrations of N-methylhistamine were detected only in lung, intestine and kidney, while in all other organs it was present only in minor quantities. HPLC-mass spectrometry is a useful method for the highly sensitive and simultaneous detection of histamine and N-methylhistamine. Histamine is present in virtually all organs, not only in those traditionally associated with histamine-mediated disease. Highest concentrations are found in stomach, lymph node, and thymus; medium concentrations in heart, spleen, and kidney; and lowest concentrations detected in intestine, brain, liver, and lung.

Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 x BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 x BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.

The Bw Cells, a Novel B Cell Population Conserved in the Whole Genus Mus

In common laboratory mouse strains, which are derived from the crossing between three subspecies, peritoneal B cells are enriched in B-1a cells characterized by the CD5(+)Mac-1(+)B220(low)IgM(high)IgD(low)CD43(+)CD9(+) phenotype. Intriguingly in other vertebrates, CD5(+)Mac-1(+) cells have never been found in a specific anatomic site. To ascertain the peculiarity of the CD5(+) peritoneal B cells in laboratory mice, we analyzed the peritoneal B cell subsets in 9 inbred and 39 outbred wild-derived mouse strains belonging to 13 different species/subspecies. We found that most of these strains do not have the CD5(+) B-1a cell population. However, all of these strains including classical laboratory mouse strains, have variable proportions of a novel B cell population: Bw, which is characterized by a unique phenotype (CD5(-)Mac-1(+)B220(high)IgM(high)IgD(high)CD43(-)CD9(-)) and is not restricted to the peritoneal cavity. Bw cells are also distinct from both B-1 and B-2 cells from a functional point of view both by proliferative responses, cytokine secretion and Ab synthesis. Moreover, transfer experiments show that bone marrow and fetal liver cells from wild mice can give rise to Bw cells in alymphoid mice. The conservation of this B cell population, but not of the CD5(+) B-1a, during evolution of the genus Mus, its readiness to respond to TLR ligands and to produce high concentration of autoantibodies suggest that Bw cells play a key role in innate immunity.