Mouse Models Of Human Disease Research Articles

A novel Non-rodent animal model of hydrochloric acid-induced acute and chronic lung injury

Hydrochloric acid is one of the most prevalent and hazardous chemicals. Accidental spills occur in industrial plants or during transportation. Exposure to HCl can induce severe health impairment, including acute and chronic pulmonary diseases. We have previously described the molecular, structural, and functional aspects of the development of chronic lung injury and pulmonary fibrosis caused by intratracheal instillation of HCl in mice. Although mouse models of human disease have many advantages, rodents are evolutionary far from human and exhibit significant anatomical and physiological differences. Genetic and anatomic similarities between rabbits and humans are significantly higher. Rabbit models of HCl-induced lung injury have been used sparsely to evaluate acute lung injury. In this study, for the first time, we utilized rabbits as a model of HCl-induced pulmonary fibrosis and chronic lung injury. We present molecular, histological, and functional evidence that demonstrate the utility of using this model for studying new pharmaceutics against pulmonary fibrosis.

A Novel Non-Rodent Animal Model of Hydrochloric Acid-Induced acute and chronic lung injury.

Hydrochloric acid is one of the most prevalent and dangerous chemicals. Accidental spills occur in industrial plants or during transportation. Exposure to HCl can induce severe health impairment, including acute and chronic pulmonary diseases. We have previously described the molecular, structural, and functional aspects of the development of chronic lung injury and pulmonary fibrosis caused by intratracheal instillation of HCl in mice. Although mouse models of human disease have many advantages, rodents are evolutionary far from human and exhibit significant anatomical and physiological differences. Genetic and anatomic similarities between rabbits and humans are significantly higher. Rabbit models of HCl-induced lung injury have been used sparsely to evaluate acute lung injury. In this study, for the first time, we utilized rabbits as a model of HCl-induced pulmonary fibrosis and chronic lung injury. We present molecular, histological, and functional evidence that demonstrate the utility of using this model for studying new pharmaceutics against pulmonary fibrosis.

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

Abstract 6806: Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden

Abstract Introduction: Neutrophils are a highly heterogenous, plastic cell population, known to play a vital role in the tumor microenvironment and progression of CMS4 colorectal cancer (CRC) metastases. We have performed a comprehensive examination of different neutrophil transcriptional states, through utilization of single-cell RNA sequencing of publicly available and independently generated data across health and malignancy in human and murine disease, and validated findings using in vivo and in vitro models of CMS4 subtype CRC. Methods: Publicly available and independently generated breast, lung and CRC datasets from human disease mouse models were integrated to establish and validate reproducibility of neutrophil-gene signatures, and pseudo-time analysis was used to identify genes driving neutrophil development along a lineage from health to tumor using Seurat on R. Findings were validated in healthy and KrasG12D/+, Trp53fl/fl, Rosa26N1CD/+ (KPN) tumor-bearing wild type or Mrp8CreCxcr2fl/fl genetically engineered mouse models with neutrophil-specific loss of Cxcr2. Results: We have identified distinct neutrophil subtypes in health and primary tumors, preserved in murine and human cancer and across multiple tumor types. In CRC metastases, discrete neutrophil transcriptomic subtypes are identified, with phenotypes from health to those identified in metastases driven by the IL-1β/CXCL8/CXCR2 signalling axis. Assessment of global signalling of single-cell transcripts has identified CD4+ T cells and macrophages as dominant regulators of the metastatic niche. Neutrophils sorted from the metastatic niche strongly suppress T cell proliferation, identifying them as immunosuppressive. Loss of neutrophil-specific Cxcr2 attenuates suppression of T cell proliferation in co-culture with neutrophils from the metastatic niche of tumor-bearing mice. T cell proliferation is unaffected in co-culture with healthy wild type or Cxcr2fl/fl neutrophils. We currently are running orthotopic intrasplenic models to assess the importance of loss of neutrophil-specific Cxcr2 in generation of metastases in vivo in CMS4 CRC. Conclusions: We speculate that ablation of metastasis specific neutrophil subsets in CMS4 CRC in combination with other therapies, may alter the immunosuppressive nature of the immune microenvironment and allow for reduction in tumor burden in metastatic disease. Citation Format: Alistair S. McLaren, Rana Fetit, Mark White, Megan L. Mills, Tamsin Lannagan, John Falconer, Xabier Cortes-Lavaud, Kathryn Gilroy, Rachel Ridgway, Colin Nixon, Varushka Naiker, Renee Njunge, Cassie J. Clarke, Declan Whyte, Kristina Kirschner, Rene Jackstadt, Jim C. Norman, Leo M. Carlin, Andrew D. Campbell, Owen J. Sansom, Colin W. Steele. Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6806.

Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety

Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.

Phosphorylation of hnRNP A1-Serine 199 Is Not Required for T Cell Differentiation and Function.

hnRNP A1 is an important RNA-binding protein that influences many stages of RNA processing, including transcription, alternative splicing, mRNA nuclear export, and RNA stability. However, the role of hnRNP A1 in immune cells, specifically CD4+ T cells, remains unclear. We previously showed that Akt phosphorylation of hnRNP A1 was dependent on TCR signal strength and was associated with Treg differentiation. To explore the impact of hnRNP A1 phosphorylation by Akt on CD4+ T cell differentiation, our laboratory generated a mutant mouse model, hnRNP A1-S199A (A1-MUT) in which the major Akt phosphorylation site on hnRNP A1 was mutated to alanine using CRISPR Cas9 technology. Immune profiling of A1-MUT mice revealed changes in the numbers of Tregs in the mesenteric lymph node. We found no significant differences in naive CD4+ T cell differentiation into Th1, Th2, Th17, or T regulatory cells (Tregs) invitro. In vivo, Treg differentiation assays using OTII-A1-Mut CD4+ T cells exposed to OVA food revealed migration and homing defects in the A1-MUT but no change in Treg induction. A1-MUT mice were immunized with NP- keyhole limpet hemocyanin, and normal germinal center development, normal numbers of NP-specific B cells, and no change in Tfh numbers were observed. In conclusion, Akt phosphorylation of hnRNP A1 S199 does not play a role in CD4+ T cell fate or function in the models tested. This hnRNP A1-S199A mouse model should be a valuable tool to study the role of Akt phosphorylation of hnRNP A1-S199 in different cell types or other mouse models of human disease.

Uma história com duas versões: efeito da polpa da raiz de yacon na asma experimental em biotérios distintos

ABSTRACT: The intestinal microbiota has an important role in animal health. Therefore, prebiotics have gained interest in the scientific community for their role in manipulating populations of intestinal microorganisms. Among prebiotics, there is Smallanthus sonchifolius Poepp. Endl (yacon) root, which can be ingested in natura or in different forms, such as flours and extracts. This prebiotic has been associated with beneficial effects in different diseases, including metabolic (like type 2 diabetes) and immunological disorders. Thus, mouse models of human diseases caused by immunological factors have been used to better understand the prebiotic effects of yacon. Since prebiotics interfere with animal microbiota, it is important to take into consideration the quality level of mouse facilities. In this way, the beneficial effects of a yacon root pulp were tested in a mouse model of allergic asthma and, considering how animal facility conditions could affect those results, we performed experiments both in conventional facilities and specific pathogen-free (SPF) ones. Our results showed that better prebiotic effects were observed in a SPF facility than in a conventional one and, in some cases, evidence of yacon beneficial effects was observed exclusively in a SPF facility. For example, there were reductions of 63.6% and 58.0% in IgE and eosinophils, respectively, in immunized/yacon-treated animals compared to immunized mice only. Taken together, our results not only showed the beneficial effects of yacon root pulp in an asthma model but also demonstrated the relevance of performing experiments with prebiotics in SPF facilities.

A Primer for Research Scientists on Assessing Mouse Gross and Histopathology Images in the Biomedical Literature.

Advances in genomic technologies have enabled the development of abundant mouse models of human disease, requiring accurate phenotyping to elucidate the consequences of genetic manipulation. Anatomic pathology, an important component of the mouse phenotyping pipeline, is ideally performed by human or veterinary pathologists; however, due to insufficient numbers of pathologists qualified to assess these mouse models morphologically, research scientists may perform "do-it-yourself" pathology, resulting in diagnostic error. In the biomedical literature, pathology data is commonly presented as images of tissue sections, stained with either hematoxylin and eosin or antibodies via immunohistochemistry, accompanied by a figure legend. Data presented in such images and figure legends may contain inaccuracies. Furthermore, there is limited guidance for non-pathologist research scientists concerning the elements required in an ideal pathology image and figure legend in a research publication. In this overview, the components of an ideal pathology image and figure legend are outlined and comprise image quality, image composition, and image interpretation. Background knowledge is important for producing accurate pathology images and critically assessing these images in the literature. This foundational knowledge includes understanding relevant human and mouse anatomy and histology and, for cancer researchers, an understanding of human and mouse tumor classification and morphology, mouse stain background lesions, and tissue processing artifacts. Accurate interpretation of immunohistochemistry is also vitally important and is detailed with emphasis on the requirement for tissue controls and the distribution, intensity, and intracellular location of staining. Common pitfalls in immunohistochemistry interpretation are outlined, and a checklist of questions is provided by which any pathology image may be critically examined. Collaboration with pathologist colleagues is encouraged. This overview aims to equip researchers to critically assess the quality and accuracy of pathology images in the literature to improve the reliability and reproducibility of published pathology data. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Targeting the biology of aging with mTOR inhibitors.

Inhibition of the protein kinase mechanistic target of rapamycin (mTOR) with the Food and Drug Administration (FDA)-approved therapeutic rapamycin promotes health and longevity in diverse model organisms. More recently, specific inhibition of mTORC1 to treat aging-related conditions has become the goal of basic and translational scientists, clinicians and biotechnology companies. Here, we review the effects of rapamycin on the longevity and survival of both wild-type mice and mouse models of human diseases. We discuss recent clinical trials that have explored whether existing mTOR inhibitors can safely prevent, delay or treat multiple diseases of aging. Finally, we discuss how new molecules may provide routes to the safer and more selective inhibition of mTOR complex 1 (mTORC1) in the decade ahead. We conclude by discussing what work remains to be done and the questions that will need to be addressed to make mTOR inhibitors part of the standard of care for diseases of aging.

Abstract 1292: CX3CR1 surface expression and phenotypic plasticity in metastasis-initiating cells

Abstract Metastasis-initiating cells (MICs) display stem cell-like features and cause metastatic recurrences leading to patients' demise. We found that prostate and breast cancer cells with high surface expression of the chemokine receptor CX3CR1 (CX3CR1high) account for ~5-7% of the whole population and are endowed with metastasis-initiating ability and exhibits stemness features, including expression of pluripotency genes Nanog and Oct4a. Notably, impairing CX3CR1 signaling hinders metastatic initiation and progression in mouse models of human disease. We also found that cancer cells lacking surface CX3CR1 expression (CX3CR1low) can re-acquire CX3CR1-associated features over time by undergoing phenotypic plasticity. Accordingly, when grafted as pure CX3CR1low populations these cells could generate disseminated tumors in mice, implying that MICs can emerge from cancer cells lacking stemness features. In vitro studies by flow cytometry show that cell surface re-exposure of CX3CR1 by CX3CR1low cells begins within 2 days post-sorting, with the whole population reaching phenotypic equilibrium at 12-16 days post-sorting. Further mechanistic studies revealed that both CX3CR1 protein trafficking and transcriptional regulation play a role in phenotypic plasticity. Additionally, disseminated tumors in mice harbor higher percentages of CX3CR1high cells than in vitro cultures, suggesting that the tissue microenvironment of target organs promotes phenotypic plasticity. These findings nominate CX3CR1 as a novel marker of MICs and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation and progression. Citation Format: Jieyi Zhang, Yetunde Oyende, Jennifer Lynch, Olimpia Meucci, Alessandro Fatatis. CX3CR1 surface expression and phenotypic plasticity in metastasis-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1292.

Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications.

Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are widely accepted because of their diversity and enormous potential for targeted genomic modifications in eukaryotes and other animals. Moreover, rapid advances in genome editing tools have accelerated the ability to produce various genetically modified animal models for studying human diseases. Given the advances in gene editing tools, these animal models are gradually evolving toward mimicking human diseases through the introduction of human pathogenic mutations in their genome rather than the conventional gene knockout. In the present review, we summarize the current progress in and discuss the prospects for developing mouse models of human diseases and their therapeutic applications based on advances in the study of programmable nucleases.

Differences in Tumor Growth and Differentiation in NSG and Humanized-BLT Mice; Analysis of Human vs. Humanized-BLT-Derived NK Expansion and Functions.

There is significant interest and debate regarding the best mouse model of human disease, since studies in wild-type mice may not always recapitulate human diseases. The NSG mouse model has been one of the most commonly used mouse models to study cancer; however, this mouse model, even though it has several advantages in regard to the ease of tumor implantation and financial feasibility, does not represent human disease due to the immunodeficient nature of this model. In this study, we performed oral and pancreatic tumor studies in NSG and hu-BLT mice and found several distinguishing features that make hu-BLT model more suitable for studying human cancer. In addition, we compared the immune function of humans to hu-BLT mice to understand the differences and similarities of the models. Oral and pancreatic cancer stem cells were implanted in NSG and hu-BLT mice. Both tumors grew robustly in NSG mice and killed them within a short period of time. On the contrary, unlike NSG mice, tumor-bearing hu-BLT mice survived longer, grew smaller tumors, and the grown tumors exhibited lower rates of expansion, with a higher surface expression of MHC-class I and lower NK cell-mediated cytotoxicity that was previously shown to have more of a differentiated phenotype. Although the peripheral blood of hu-BLT mice in comparison to that of humans had lower percentages of NK cells and cytotoxic function, it mediated a higher secretion of IFN-γ, likely contributing to the differentiation of the tumor cells and subsequent decrease in the tumor size in the hu-BLT mice in comparison to the NSG mice. Spleen-derived hu-BLT mouse NK cells were able to expand in the presence of autologous osteoclasts and substantially increase both cytotoxicity and secretion of IFN-γ, similar to those seen in peripheral blood-derived human NK cells, indicating that NK cells from hu-BLT mice are capable of expansion and functional activation when activating signals are given. Thus, the many similarities between human and hu-BLT mouse immune systems make this mouse model more appropriate to study human cancer. In particular, it is well-suited for studies of allogeneic NK cell-based immunotherapy in cancer treatment. The advantages and challenges of hu-BLT mice in cancer studies are also discussed in this report.

Targeted RNA editing in brainstem alleviates respiratory dysfunction in a mouse model of Rett syndrome

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editasewt) and either Mecp2-targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2-targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2-targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

Rhein Inhibits PI3K/Akt/mTOR Signaling Pathway to Show Efficacy in an Ulcerative Colitis Mice Model

Rhein Inhibits PI3K/Akt/mTOR Signaling Pathway to Show Efficacy in an Ulcerative Colitis Mice Model

Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days.

Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell culture methods are suboptimal for effectively testing new therapies and live mouse models are expensive, time consuming and require invasive procedures. Fetal organ culture, organoids, microfluidics and culture of thick sections of adult organs all aim to fill the knowledge gap between monolayer culture and live mouse studies. Here we report on an ex vivo organ perfusion system that can support whole adult mouse organs. Ex vivo perfusion of healthy and diseased mouse organs allows for real-time analysis that provides immediate feedback and accurate data collection throughout the experiment. Having a suitable normothermic ex vivo perfusion system for mouse organs provides a tool that will help contribute to our understanding of kidney physiology and disease and can take advantage of the many mouse models of human disease that already exist. Furthermore, an ex vivo kidney perfusion system can be used for testing novel cell therapies, drug screening, drug validation and for the detection of nephrotoxic substances. Critical to the success of mouse ex vivo organ perfusion is having a suitable bioreactor to maintain the organ. Here we have focused on the mouse kidney and mathematically modeled, built and validated a bioreactor that can maintain a kidney for 7 days. The long duration of the ex vivo perfusion will help to advance studies on kidney disease and can rapidly test for new regenerative medicine therapies compared to whole animal studies.

Mapping Brain‐wide Pathologies in Mouse Models of Neurodegeneration

Neurodegenerative diseases are characterized by deposition of abnormal protein aggregates in the brain in predictable spatial and temporal patterns. This selective vulnerability to pathology, which encompasses vulnerabilities of cell types, regions, and brain networks, is a fundamental feature of disease that is not yet understood. Mouse models of human disease are required for preclinical investigations into molecular and cellular mechanisms underlying vulnerability and resilience. Yet, little data exists on how well aligned commonly used models are with the brain‐wide human spatiotemporal vulnerability maps. To address this gap and more deeply characterize existing models, we built a high‐throughput whole brain imaging and informatics pipeline to systematically quantify spatiotemporal patterns of disease‐relevant pathologies in mice. Using whole brain clearing, antibody labeling, and lightsheet imaging, we have quantified tyrosine hydroxylase levels in a mouse model of Parkinson’s Disease with nigrostriatal degeneration and show the utility of 3D imaging to identify disease‐relevant regional vulnerabilities.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis

Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures—weight loss and intestinal pathology—showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.