Common Inbred Strains Research Articles

Validation of novel conditional ligands and large-scale detection of antigen-specific T cells for H-2Dd and H-2Kd

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2Dd and H2Kd. In addition, we demonstrate the ability of the three H2d molecules and two additional C57BL/6 H2b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases.

Sex differences in avoidance behavior and cued threat memory dynamics in mice: Interactions between estrous cycle and genetic background

Anxiety disorders are the most prevalent mental illnesses worldwide, exhibit high heritability, and affect twice as many women as men. To evaluate potential interactions between genetic background and cycling ovarian hormones on sex differences in susceptibility to negative valence behaviors relevant to anxiety disorders, we assayed avoidance behavior and cued threat memory dynamics in gonadally-intact adult male and female mice across four common inbred mouse strains: C57Bl/6J, 129S1/SVlmJ, DBA/2J, and BALB/cJ. Independent of sex, C57Bl/6J mice exhibited low avoidance but high threat memory, 129S1/SvlmJ mice high avoidance and high threat memory, DBA/2J mice low avoidance and low threat memory, and BALB/cJ mice high avoidance but low threat memory. Within-strain comparisons revealed reduced avoidance behavior in the high hormone phase of the estrous cycle (proestrus) compared to all other estrous phases in all strains except DBA/2J, which did not exhibit cycle-dependent behavioral fluctuations. Robust and opposing sex differences in threat conditioning and extinction training were found in the C57Bl/6J and 129S1/SvlmJ lines, whereas no sex differences were observed in the DBA/2J or BALB/cJ lines. C57Bl/6J males exhibited enhanced acute threat memory, whereas 129S1/SvlmJ females exhibited enhanced sustained threat memory, compared to their sex-matched littermates. These effects were not mediated by estrous cycle stage or sex differences in active versus passive defensive behavioral responses. Our data demonstrate that core features of behavioral endophenotypes relevant to anxiety disorders, such as avoidance and threat memory, are genetically driven yet dissociable and can be influenced further by cycling ovarian hormones.

A universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation.

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Comparison of three common inbred mouse strains reveals substantial differences in hippocampal GABAergic interneuron populations and in vitro network oscillations.

A major challenge in neuroscience is to pinpoint neurobiological correlates of specific cognitive and neuropsychiatric traits. At the mesoscopic level, promising candidates for establishing such connections are brain oscillations that can be robustly recorded as local field potentials with varying frequencies in the hippocampus in vivo and in vitro. Inbred mouse strains show natural variation in hippocampal synaptic plasticity (e.g. long-term potentiation), a cellular correlate of learning and memory. However, their diversity in expression of different types of hippocampal network oscillations has not been fully explored. Here, we investigated hippocampal network oscillations in three widely used inbred mouse strains: C57BL/6J (B6J), C57BL/6NCrl (B6N) and 129S2/SvPasCrl (129) with the aim to identify common oscillatory characteristics in inbred mouse strains that show aberrant emotional/cognitive behaviour (B6N and 129) and compare them to "control" B6J strain. First, we detected higher gamma oscillation power in the hippocampal CA3 of both B6N and 129 strains. Second, higher incidence of hippocampal sharp wave-ripple (SPW-R) transients was evident in these strains. Third, we observed prominent differences in the densities of distinct interneuron types and CA3 associative network activity, which are indispensable for sustainment of mesoscopic network oscillations. Together, these results add further evidence to profound physiological differences among inbred mouse strains commonly used in neuroscience research.

Mouse strain-dependent variation in metabolic associated fatty liver disease (MAFLD): a comprehensive resource tool for pre-clinical studies

Non-alcoholic steatohepatitis (NASH), characterized as the joint presence of steatosis, hepatocellular ballooning and lobular inflammation, and liver fibrosis are strong contributors to liver-related and overall mortality. Despite the high global prevalence of NASH and the substantial healthcare burden, there are currently no FDA-approved therapies for preventing or reversing NASH and/or liver fibrosis. Importantly, despite nearly 200 pharmacotherapies in different phases of pre-clinical and clinical assessment, most therapeutic approaches that succeed from pre-clinical rodent models to the clinical stage fail in subsequent Phase I-III trials. In this respect, one major weakness is the lack of adequate mouse models of NASH that also show metabolic comorbidities commonly observed in NASH patients, including obesity, type 2 diabetes and dyslipidaemia. This study provides an in-depth comparison of NASH pathology and deep metabolic profiling in eight common inbred mouse strains (A/J, BALB/c, C3H/HeJ, C57BL/6J, CBA/CaH, DBA/2J, FVB/N and NOD/ShiLtJ) fed a western-style diet enriched in fat, sucrose, fructose and cholesterol for eight months. Combined analysis of histopathology and hepatic lipid metabolism, as well as measures of obesity, glycaemic control and insulin sensitivity, dyslipidaemia, adipose tissue lipolysis, systemic inflammation and whole-body energy metabolism points to the FVB/N mouse strain as the most adequate diet-induced mouse model for the recapitulation of metabolic (dysfunction) associated fatty liver disease (MAFLD) and NASH. With efforts in the pharmaceutical industry now focussed on developing multi-faceted therapies; that is, therapies that improve NASH and/or liver fibrosis, and concomitantly treat other metabolic comorbidities, this mouse model is ideally suited for such pre-clinical use.

Trans-driven variation in expression is common among detoxification genes in the extreme generalist herbivore Tetranychus urticae.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T. urticae. Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness as is commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis, with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans-driven up- and downregulation. In particular, multiple CYPs and DOGs with broad substrate-specificities for pesticides or plant specialized compounds were exceptionally highly upregulated as a result of trans-regulatory variation, or in some cases synergism of cis and trans, in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans-driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T. urticae, and also suggests modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects.

Two Loci Contribute to Age-Related Hearing Loss Resistance in the Japanese Wild-Derived Inbred MSM/Ms Mice

An MSM/Ms strain was established using Japanese wild mice, which exhibit resistance to several phenotypes associated with aging, such as obesity, inflammation, and tumorigenesis, compared to common inbred mouse strains. MSM/Ms strain is resistant to age-related hearing loss, and their auditory abilities are sustained for long durations. The age-related hearing loss 3 (ahl3) locus contributes to age-related hearing in MSM/Ms strain. We generated ahl3 congenic strains by transferring a genomic region on chromosome 17 from MSM/Ms mice into C57BL/6J mice. Although C57BL/6J mice develop age-related hearing loss because of the ahl allele of the cadherin 23 gene, the development of middle- to high-frequency hearing loss was significantly delayed in an ahl3 congenic strain. Moreover, the novel age-related hearing loss 10 (ahl10) locus associated with age-related hearing resistance in MSM/Ms strain was mapped to chromosome 12. Although the resistance effects in ahl10 congenic strain were slightly weaker than those in ahl3 congenic strain, slow progression of age-related hearing loss was confirmed in ahl10 congenic strain despite harboring the ahl allele of cadherin 23. These results suggest that causative genes and polymorphisms of the ahl3 and ahl10 loci are important targets for the prevention and treatment of age-related hearing loss.

Independent and Interactive Effects of Genetic Background and Sex on Tissue Metabolomes of Adipose, Skeletal Muscle, and Liver in Mice.

Genetics play an important role in the development of metabolic diseases. However, the relative influence of genetic variation on metabolism is not well defined, particularly in tissues, where metabolic dysfunction that leads to disease occurs. We used inbred strains of laboratory mice to evaluate the impact of genetic variation on the metabolomes of tissues that play central roles in metabolic diseases. We chose a set of four common inbred strains that have different levels of susceptibility to obesity, insulin resistance, and other common metabolic disorders. At the ages used, and under standard husbandry conditions, these lines are not overtly diseased. Using global metabolomics profiling, we evaluated water-soluble metabolites in liver, skeletal muscle, and adipose from A/J, C57BL/6J, FVB/NJ, and NOD/ShiLtJ mice fed a standard mouse chow diet. We included both males and females to assess the relative influence of strain, sex, and strain-by-sex interactions on metabolomes. The mice were also phenotyped for systems level traits related to metabolism and energy expenditure. Strain explained more variation in the metabolite profile than did sex or its interaction with strain across each of the tissues, especially in liver. Purine and pyrimidine metabolism and pathways related to amino acid metabolism were identified as pathways that discriminated strains across all three tissues. Based on the results from ANOVA, sex and sex-by-strain interaction had modest influence on metabolomes relative to strain, suggesting that the tissue metabolome remains largely stable across sexes consuming the same diet. Our data indicate that genetic variation exerts a fundamental influence on tissue metabolism.

ICLAS LAQ Network for the Promotion of Animal Quality in Research.

ICLAS Laboratory Animal Quality Network (LAQN) programs currently consist of the Performance Evaluation Program (PEP), which focuses on microbial monitoring by and for laboratory animal diagnostic laboratories, and the Genetic Reference Monitoring Program (GENRef), which provides assay-ready reference DNA for genetic testing of mouse strains. Since 2008, PEP has grown to become a truly international program with participating laboratories in 5 continents. Launched in 2016, GENRef currently distributes DNA from 12 common inbred mouse strains for use in genetic monitoring of locally inbred colonies as well as for genetic testing of stocks, particularly genetically engineered stocks, of uncertain origins. GENRef has the capacity to include additional strains as well as additional species. PEP and GENRef provide the reagents at cost, as a resource to the international scientific community, in the interest of improving research quality in an environment of growing concern for research quality, rigor, and reproducibility.

Identification of Basp1 as a novel angiogenesis-regulating gene by multi-model system studies.

We have previously used the genetic diversity available in common inbred mouse strains to identify quantitative trait loci (QTLs) responsible for the differences in angiogenic response using the corneal micropocket neovascularization (CoNV) assay. Employing a mouse genome-wide association study (GWAS) approach, the region on chromosome 15 containing Basp1 was identified as being significantly associated with angiogenesis in inbred strains. Here, we developed a unique strategy to determine and verify the role of BASP1 in angiogenic pathways. Basp1 expression in cornea had a strong correlation with a haplotype shared by mouse strains with varied angiogenic phenotypes. In addition, inhibition of BASP1 demonstrated a dosage-dependent effect in both primary mouse brain endothelial and human microvascular endothelial cell (HMVEC) migration. To investigate its role in vivo, we knocked out basp1 in transgenic kdrl:zsGreen zebrafish embryos using a widely adopted CRISPR-Cas9 system. These embryos had severely disrupted vessel formation compared to control siblings. We further show that basp1 promotes angiogenesis by upregulating β-catenin gene and the Dll4/Notch1 signaling pathway. These results, to the best of our knowledge, provide the first in vivo evidence to indicate the role of Basp1 as an angiogenesis-regulating gene and opens the potential therapeutic avenues for a wide variety of systemic angiogenesis-dependent diseases.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1 -/-, Cybb -/- or Casp1 -/-/Cybb -/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

The Japanese Wild-Derived Inbred Mouse Strain, MSM/Ms in Cancer Research.

Simple SummarySince human studies on tumor susceptibility alleles require vast numbers of DNA samples from both cancer patients and well-matched controls, their investigation and identification in humans have been complemented using mouse models. However, a number of confounding factors are associated with this type of research, including heterogeneity, weak genetic interactions, and lifestyle habits. Inbred strains relatively recently established from wild mice are often more resistant to carcinogenic stimulation and various pathogens than standard inbred mouse strains. A Japanese wild-derived inbred mouse strain, MSM/Ms has been used to map tumor resistance loci as well as other Quantitative Trait Loci (QTL) in Japan. Furthermore, genetic tools have been developed with MSM/Ms. MSM/Ms genomic sequences are currently available, which have greatly promoted the identification of tumor resistance loci as well as genes controlling quantitative variations and provide a more detailed understanding of gene function. MSM/Ms is a unique inbred mouse strain derived from the Japanese wild mouse, Mus musculus molossinus, which has been approximately 1 million years genetically distant from standard inbred mouse strains mainly derived from M. m. domesticus. Due to its genetic divergence, MSM/Ms has been broadly used in linkage studies. A bacterial artificial chromosome (BAC) library was constructed for the MSM/Ms genome, and sequence analysis of the MSM/Ms genome showed approximately 1% of nucleotides differed from those in the commonly used inbred mouse strain, C57BL/6J. Therefore, MSM/Ms mice are thought to be useful for functional genome studies. MSM/Ms mice show unique characteristics of phenotypes, including its smaller body size, resistance to high-fat-diet-induced diabetes, high locomotive activity, and resistance to age-onset hearing loss, inflammation, and tumorigenesis, which are distinct from those of common inbred mouse strains. Furthermore, ES (Embryonic Stem) cell lines established from MSM/Ms allow the MSM/Ms genome to be genetically manipulated. Therefore, genomic and phenotypic analyses of MSM/Ms reveal novel insights into gene functions that were previously not obtained from research on common laboratory strains. Tumorigenesis-related MSM/Ms-specific genetic traits have been intensively investigated in Japan. Furthermore, radiation-induced thymic lymphomas and chemically-induced skin tumors have been extensively examined using MSM/Ms.

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.

FLT3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation.

Conventional dendritic cells (cDCs) are comprised of two major subsets, type 1 cDC (cDC1) and type 2 cDC (cDC2). As each cDC subset differentially influences the nature of immune responses, we sought factors that would allow the manipulation of their relative abundance. Notably, cDC1 are less abundant than cDC2 in both lymphoid and nonlymphoid organs. We demonstrate that this bias is already apparent in bone marrow precommitted precursors. However, comparison of five common inbred strains revealed a disparity in precursor-product relationship, in which mice with fewer precursors to cDC1 had more cDC1. This disparity associated with contrasting variations in CD135 (FLT3) expression on cDC subsets. Hence, we characterized the response to FLT3 ligand during cDC1 and cDC2 lineage differentiation and find that although FLT3 ligand is required throughout cDC2 differentiation, it is surprisingly dispensable during late-stage cDC1 differentiation. Overall, we find that tight regulation of FLT3 ligand levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

Angiogenic responses in a 3D micro-engineered environment of primary endothelial cells and pericytes.

Angiogenesis plays a key role in the pathology of diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. Understanding the driving forces of endothelial cell migration and organization, as well as the time frame of these processes, can elucidate mechanisms of action of important pathological pathways. Herein, we have developed an organ-specific microfluidic platform recapitulating the in vivo angiogenic microenvironment by co-culturing mouse primary brain endothelial cells with brain pericytes in a three-dimensional (3D) collagen scaffold. As a proof of concept, we show that this model can be used for studying the angiogenic process and further comparing the angiogenic properties between two different common inbred mouse strains, C57BL/6J and 129S1/SvlmJ. We further show that the newly discovered angiogenesis-regulating gene Padi2 promotes angiogenesis through Dll4/Notch1 signaling by an on-chip mechanistic study. Analysis of the interplay between primary endothelial cells and pericytes in a 3D microfluidic environment assists in the elucidation of the angiogenic response.

Comparison of Metabolomic Profiles of Organs in Mice of Different Strains Based on SPME-LC-HRMS.

Given that the extent to which genetics alters the metabolomic profile of tissues is still poorly understood, the current study aimed to characterize and investigate the metabolite profiles of brain, liver, kidney and skeletal muscle of two common mouse inbred strains (BALB/c, C57BL/6) and one outbred stock (CD1) for strain-specific differences. Male mice (n = 15) at the age of 12 weeks were used: BALB/c (n = 5), C57BL/6 (n = 5) and CD1 (n = 5). Solid phase microextraction (SPME) was applied for the extraction of analytes from the tissues. SPME fibers (approximately 0.2 mm in diameter) coated with a biocompatible sorbent (4 mm length of hydrophilic-lipophilic balanced particles) were inserted into each organ immediately after euthanasia. Samples were analyzed using liquid chromatography coupled to a Q-Exactive Focus Orbitrap mass spectrometer. Distinct interstrain differences in the metabolomic patterns of brain and liver tissue were revealed. The metabolome of kidney and muscle tissue in BALB/c mice differed greatly from C57BL/6 and CD1 strains. The main compounds differentiating all the targeted organs were alpha-amino acids, purine nucleotides and fatty acid esters. The results of the study indicate that the baseline metabolome of organs, as well as different metabolic pathways, vary widely among general-purpose models of laboratory mice commonly used in biomedical research.

Adaptation of retinal ganglion cell function during flickering light in the mouse

Rapid dilation of retinal vessels in response to flickering light (functional hyperemia) is a well-known autoregulatory response driven by increased neural activity in the inner retina. Little is known about flicker-induced changes of activity of retinal neurons themselves. We non-invasively investigated flicker-induced changes of retinal ganglion cell (RGC) function in common inbred mouse strains using the pattern electroretinogram (PERG), a sensitive measure of RGC function. Flicker was superimposed on the pattern stimulus at frequencies that did not generate measurable flicker-ERG and alter the PERG response. Transition from flicker at 101 Hz (control) to flicker at 11 Hz (test) at constant mean luminance induced a slow reduction of PERG amplitude to a minimum (39% loss in C57BL/6J mice and 52% loss in DBA/2J mice) 4–5 minutes after 11 Hz flicker onset, followed by a slow recovery to baseline over 20 minutes. Results demonstrate that the magnitude and temporal dynamics of RGC response induced by flicker at 11 Hz can be non-invasively assessed with PERG in the mouse. This allows investigating the functional phenotype of different mouse strains as well as pathological changes in glaucoma and optic nerve disease. The non-contact flicker-PERG method opens the possibility of combined assessment of neural and vascular response dynamics.

Effects of Soy in Laboratory Rodent Diets on the Basal, Affective, and Cognitive Behavior of C57BL/6 Mice.

Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Animal Modeling for Hologenome: New inbred and conplastic rat exercise models for uncovering crosstalk between nuclear DNA, mitochondrial DNA, and gut microbiota

IntroductionThe hologenome is an ensemble of nuclear DNA (nDNA), mitochondrial DNA (mtDNA), and the microbiome that forms a unit of genetic selection that can propagate from one generation to the next. There is increasing evidence for inherited mitochondria‐gut microbiota involvement in cardiovascular and metabolic diseases. Here, we expand on the selectively‐bred Low Capacity Runner/High Capacity Runner (LCR/HCR) rat exercise model system into a genetically defined rat model framework for studying holobionts in controlled laboratory settings.MethodsAt Generation 23 of selection, we identified extreme representatives of female and male LCR and HCR rats and conducted a series of brother‐sister mating to produce inbred strains‐ LCR/Tol and HCR/Tol. We also developed two conplastic strains, one with nDNA from HCR and mtDNA from LCR, and the other with nDNA from LCR and mtDNA from HCR ‐LCR.HCRmt/Tol and HCR.LCRmt/Tol. mtDNA from LCR/Tol and HCR/Tol strains were compared with publicly available mtDNA from common inbred strains. Rats were phenotyped for cardiovascular risks, which included treadmill exercise capacity, VO2max, echocardiography, and blood pressure (BP) telemetry. Fecal microbial communities were profiled using 16S rRNA sequencing and metatranscriptomics.ResultsAs expected, HCR/Tol had significantly higher exercise capacity compared to LCR/Tol but little effect by mismatched mtDNA suggesting that the nDNA is dominant for regulating aerobic exercise capacity. VO2max measurement confirmed high exercise capacity in HCR/Tol and low in LCR/Tol. LCR mtDNA was identical to mtDNA from Wistar Kyoto inbred strain, while HCR mtDNA was identical to mtDNA from Fischer 344 × Brown Norway F1‐hybrid strain. Consistent with reports from selected LCR HCR rats, LCR/Tol showed significantly increased mean arterial pressure (MAP), systolic BP (SBP), and diastolic BP (DBP) compared to the HCR/Tol. Transfer of mtDNA from HCR to LCR nDNA background decreased MAP, SBP, DBP, and preserved diastolic cardiac function. In contrast, there was no difference in BP or cardiac parameters between HCR/Tol and HCR.LCRmt/Tol. Microbiome analysis indicated that host genomes co‐inhabit with differential commensal microbiota. Metatranscriptomics showed HCR/Tol had enhanced function in carbohydrate and amino acid metabolism, microbial metabolites production, and stress management.ConclusionWe successfully generated and systematically characterized genotypes and phenotypes of inbred and conplastic LCR/Tol and HCR/Tol animal models for holobiont studies. The inbred and conplastic strains retained key features of LCR HCR selectively bred models ‐‐ some influenced by nDNA‐mtDNA interaction. These models provide for a new pre‐clinical method to explore gut microbiota as therapeutic targets for complex diseases such as hypertension.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell-intrinsic regulation of peripheral memory-phenotype T cell frequencies.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.