The mouse as a model organism(1).pptx

MODEL ORGANISMS: THE MOUSE Dr Jill Johnson Learning objectives The mouse as a model organism Mouse vs. human: similarities and differences Genetic manipulation in mice Mouse models of chronic lung disease Answering immunological questions with mice Answering pharmacological questions with mice Mouse...

MODEL ORGANISMS: THE MOUSE Dr Jill Johnson Learning objectives The mouse as a model organism Mouse vs. human: similarities and differences Genetic manipulation in mice Mouse models of chronic lung disease Answering immunological questions with mice Answering pharmacological questions with mice Mouse vs. human: similarities and differences Mice share about 80% sequence homology with humans This genetic similarity, along with their small size, rapid breeding, and ease of handling, provides important advantages to scientists for practical and financial reasons Specifically, there is a high degree of similarity in transcription factor networks but a great deal of divergence in the regulatory elements that control gene transcription in the mouse and human genomes The human body is about 2500x larger than that of the mouse, but the mouse basal metabolic rate is The history of using mice in research In 1900 on a farm in Granby, Massachusetts, mousefancier Abbie Lathrop turned her hobby into a business by selling some of the animals she bred to researchers at the nearby Massachusetts Institute of Technology, pursuing the new science of genetics In 1909 one such scientist, called Clarence Cook Little, mated closely related mice for generation after generation, creating the first inbred strain In the 1970s and 1980s, spontaneous mutations in mice stimulated research into immune system disorders (e.g. SCID) Inbred mouse strains Most research studies use inbred mice to reduce variability Hundreds of inbred mouse strains have been developed The most popular in biological research are C57/Bl6 and Balb/c C57/Bl6 Balb/c Differences between strains Balb/c mice generally develop a more robust humoral immune response than C57/Bl6, making them more suitable as models of antibody-driven diseases, e.g. allergy and asthma C57/Bl6 mice develop more robust cell-mediated responses to bacteria and parasites, making them a good model for infectious diseases (Balb/c mice do not handle infection well, which is a major ethical issue) Ethics of using mice in experiments There is a lot of variation in how stringent the rules are for using experimental mice, depending on the country The UK has the strictest animal use regulations in the world – ASPA (1986) All animal experiments in the UK are overseen by the Home Office For a scientist to be qualified to work with protected experimental animals (all vertebrates + cephalopods), a personal licence must be acquired (about 1 week of training and several exams) For a research group to be allowed to work with protected animals, a project license must be in place describing all the procedures, with relevant scientific justification and expected outcomes in the Immunological differences mice vs. humans Table 1 A brief overview of the immunological differences between mice and humans Attribute Mouse Human References Proportion of leukocytes in the blood 75–90% lymphocytes 10–25% neutrophils 50–70% neutrophils 30–50% lymphocytes Costimulatory signaling 80% of CD4+ and 50% of CD8+ T cells express CD28 ICOS is not required for B cell maturation B7-H3 inhibits T cell activation 100% of CD4+ and CD8+ T cells express CD28 ICOS is required for B cell maturation and IgM production B7-H3 promotes T cell activation [15, 16] Immunoglobulin isotypes IgD, IgM, IgA, IgE, IgG1, IgG2a/c, IgG2b, IgG3 IgD, IgM, IgA1, IgA2, IgE, IgG1, IgG2, IgG3, IgG4 Immunoglobulin class switching IL-4 induces IgG1 and IgE IL-4 induces IgG4 and IgE Helper T cell differentiation IFN-α does not activate STAT4 and does not induce Th1 polarization Clear Th1/Th2 differentiation in mice IFN-α induces Th1 polarization via STAT4 Multiple T helper cell subsets occur simultaneously Responses to infection Eradication of schistosomiasis requires a Th1 response and IFN-γ Low susceptibility to Mycobacterium tuberculosis; noncaseating granulomas; no latent infection Eradication of schistosomiasis requires a Th2 response and IgE Highly susceptible to Mycobacterium tuberculosis; caseating granulomas; latent infection is common K Rydell-Törmänen, JR Johnson. The Applicability of Mouse Models to the Study of Human Disease Methods Mol Biol. 2019;1940:3-22. doi: 10.1007/978-1-4939-9086-3_1. Using mice as a tool to answer questions A number of tools are available for mouse manipulation to allow researchers to ask questions about the role/impact of specific cells under disease conditions: Adoptive transfer Knockout strains Cre/LoxP system (reporter mice) CRISPR/Cas9 system Adoptive transfer It is possible to transfer the immune system of one mouse into another by performing a bone marrow transplant known as adoptive transfer This allows for tracking different cell types, i.e. into a tumour Adoptive transfer GFP-labelled immune cells can be used to track tumour infiltration/inflammation https://www.frontiersin.org/articles/10.3389/fimmu.2020.01514/full Knockout mice Knockout mice are missing a gene, either in every cell of the body (conventional) or in only specific cells/tissues (conditional) Conditional knockout strains can also control the timing of gene knockout, i.e. only in adulthood Conventional gene knockout Targeting vectors are used to ‘reprogram’ the genome in embryonic stem (ES) cells ES clones are used to establish chimaeric embryos, which are then implanted Knockout mice are then ‘backcrossed’ onto a wildtype mouse to ensure the rest of the genome matches the parent strain (which is used as a control) Conventional gene knockout Johnson et al., Am J Physiol Lung Cell Mol Physiol 2007 Delivering HDM extract to IL-4 deficient mice led to airway inflammation, but it was milder and Th1-skewed (macrophages, IFN , IgG2a) rather than the robust Th2 response seen in wild type mice Conventional gene knockout IL-4 deficient mice do not develop airway remodelling in response to allergen administration https://journals.physiology.org/doi/full/10.1152/ajplung.00056.2007 Conditional gene knockout Some genes are necessary during development, so conventional knockout mice are not viable Gene expression can be controlled by giving the mice a drug (usually tetracycline/doxycycline or tamoxifen) in their drinking water to turn gene expression on or off in adulthood Conditional gene knockout Selectively knocking out a specific gene from a specific cell type can reveal disease mechanisms https://www.mdpi.com/2073-4409/12/12/1658 Reporter mice - the Cre-LoxP system The Cre-LoxP system was developed to turn on (or off) the expression of a certain gene in a particular cell type, i.e. GFP expression in lung epithelial cells using Cre recombinase Reporter mice - the Cre-LoxP system Gli1:Cre ERT2; Rosa26:lox-STOP-loxtdTomato mice were used to determine the cell types responsible for liver fibrosis (hint: it’s pericytes!) 0.1053/j.gastro.2020.03.075 The CRISPR/Cas9 system Multiple variations on the CRISPR/Cas9 system have been adapted to mice to produce gene edited mice The CRISPR/Cas9 system Loss of MCAM (CD146) increases the clonogenicity and regenerative capacity of mammary epithelial cells, and promotes proliferation, differentiation, and ductal morphogenesis in the mammary epithelium in knockout mice ://www.ncbi.nlm.nih.gov/pmc/articles/PMC10761817/pdf/41467_2023_Article_443 Transgenic mice Transgenic mice are genetically modified to express certain genes in particular cell types The names of these mice can be very complicated, but they provide a lot of information on how the mouse was designed Transgenic mice Transgenic mouse models of human diseases are invaluable tools for studying pathogenic mechanisms and testing interventions and therapeutics, but these models need to be validated https://www.nature.com/articles/s41597-021-01054-y Mouse models of disease Robert Hooke performed the first recorded inquirydriven experiments on mice in 1664, when he investigated the effects of changes in air pressure on respiratory function A mouse model of a human disease needs to fulfil two important features: the accuracy of its aetiology, i.e. it employs a physiologically relevant method of disease induction The accuracy of its presentation, i.e. its ability to recapitulate the features of human disease Mouse models of allergic asthma The World Health Organization (WHO) defines asthma as a chronic disease characterized by recurrent attacks of breathlessness and wheezing, which may vary in severity and frequency from person to person The disease is characterized by airway hyperresponsiveness, airway smooth muscle thickening, increased mucus secretion and collagen deposition, driven by prominent Th2 inflammation affecting both large and small airways Mouse strains differ in their susceptibility to allergic airway inflammation C57Bl/6 is generally considered a Th1-skewed strain, whereas BALB/c is regarded as a Th2-skewed strain Ovalbumin (OVA) models of asthma In ovalbumin-driven models of asthma, the mouse is first sensitized to OVA by an intraperitoneal injection also containing aluminium salts (alum) as an adjuvant to drive a Th2-polarised immune response The mouse is them exposed to an aerosol of OVA (challenge) to induce allergic inflammation in the lung This model does not reflect the route of sensitization in humans, and uses an antigen with no biological activity, i.e. no danger signal Airway remodelling does not occur due to the brief exposure time Prolonged OVA delivery induces respiratory tolerance, i.e. the inflammation subsides Poor accuracy in terms of aetiology Moderate accuracy in terms of presentation Allergen-driven models better recapitulate human asthma Good accuracy in terms of aetiology Good accuracy in terms of presentation Johnson et al., Am J Respir Crit Care Med 2004 The role of pericytes in allergic asthma Johnson et al., Am J Physiol Lung Cell Mol Physiol 2015 Prolonged HDM exposure induces EMT after about 10 weeks, but airway remodelling first appears after about 3 weeks of HDM exposure Additional processes must be at play This study investigated the role of tissue-resident mesenchymal stem cells (pericytes) in the development of airway remodelling and airway hyperresponsiveness The role of pericytes in allergic asthma Johnson et al., Am J Physiol Lung Cell Mol Physiol 2015 Genetic tracing of pericytes (DsRed-NG2) allowed us to visualise pericyte accumulation in airway smooth muscle bundles Further studies with a PDGFRβ inhibitor demonstrated that pericyte accumulation in the airway wall is proportional to the severity of airway hyperresponsiveness Impact of CXCL12 neutralization (LIT-927) Impact of CXCL12 neutralization (LIT-927) A Acclimati on *** B *** *** *** C *** F PBS + VEH PBS + LIT LIT-927 i.n. 5 d/wk HDM 5d/wk D Cull *** *** *** E * HDM + VEH HDM + LIT Bignold, Shammout et al., submitted Summary Mice share about 80% genetic homology with humans, with many similarities in terms of physiology and immunology Some obvious differences need to be acknowledged, i.e. size and metabolic rate A large number of genetic tools are available to allow for more sophisticated studies involving mice Mouse models of human disease can be very useful clinical tools, provided that the aetiology and presentation of the disease are similar Using allergic asthma as a specific example, genetic tools have been used to demonstrate the critical nature of Th2 immunity in this disease as well as the molecular and cellular mechanisms driving airway remodelling These models can also be used in preclinical testing