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Karina Vargová, and others…

Biological mode ls of diseases. block seminar. Karina Vargová, and others…. warning: neither the PDF, nor the PPT versions of this presentation are official study materials. First Medical Faculty, Institute of Pathological Physiology. Content. usage of animal models in research

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Karina Vargová, and others…

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  1. Biological models of diseases block seminar Karina Vargová, and others… warning: neither the PDF, nor the PPT versions of this presentation are official study materials First Medical Faculty, Institute of Pathological Physiology

  2. Content • usage of animal models in research • model organisms • mouse models of human diseases with - natural mutations - induced mutations - transgenic organisms

  3. Use of animals as a biological models • first reference – Greece 2nd.cent. (dissections, vivisections, experimentation) • most of our anatomical and physiological knowledge • until 70`s – experimentation also on humans (soldiers, prisoners, mentally disabled) • ethical aspects • elimination efforts (3Rs - Refine, Reduce, Replace) • cell cultures and cell lines, mathematical modeling, ... „In vivo veritas“

  4. Animal models in biomedicine • basic research of disease mechanisms • development of new therapeutic strategies • drug discovery and development • preclinical studies • testing of toxicity • development of new animal models

  5. How to choose an appropriate animal model? • Appropriate to the condition being investigated • Respond in the same way as humans to the proposed treatment(s) for the character being investigated (based on specific evidence, or it may be assumed from the biological similarity between animals and humans, the more fundamental the biological process, the more likely the animal model and humans will respond similarly) • Well designed experiments (are capable of demonstrating the response, appropriate size, length of the animal, adequate controls and amounts of animals for statistical evaluation) • Models are highly specific (may work in some study, but not in other) • Clinical study will validate the model (e.g. testing of new drug)

  6. Requirements for an ideal model organism a) Biological • accessibility, breeding possibilities in laboratory • quick development • not complicated life cycle • production of enough offspring b) Methodological • possibility of genetic manipulation (crossbreeding, spontaneous or aimed mutagenesis, reverse genetics) • size, longevity • accessibility to sequencing data • possibility of transgenesis

  7. Some typical model organisms Invertebrates: roundworm(Caenorhabditiselegans) fruit fly (Drosophila melanogaster) Vertebrates: platanna (Xenopuslaevis) zebrafish (Daniorerio) chicken (Gallus gallusdomesticus) mouse(Musmusculus) rat(Ratusnorvegicus) rabbit(Oryctolaguscuniculus) ... dog, cat, pig, monkey, primates

  8. Roundworm (Caenorhabditis elegans) • developmental biology(1974; Sydney Brenner) • genome completely sequenced – 100x106bp(1998; 2002) • strictlydefinedsomatic cell number (eutely), transparent • recently study ofmoleculargeneticsofaging • Nobel prices: 2002 - development, programmed cell death 2006 - RNA interference (A.Fire, C.C.Mello) 2008 - GFP (M.Chalfie) (S.Brenner, H.R.Horvitz, J. Sulston) (Physiology medicine) (chemistry) • Friut fly (Drosophila melanogaster) • genetic studies (T.H.Morgan, 1910) • huge amount of mutants and genetic modifications • study of genetics of heart disease, neurodegenerative diseases • pre-mammal alternative, acceleration of the therapy testing

  9. platanna (Xenopus laevis) • developmental biology, toxicology • genes mutated in human cystic kidney • disorders, action of human disease genes associated with • genetic instability and elevated cancer risk e.g. ATM, BRCA1, • or c-Myc...) • zebrafish (Danio rerio) • developmental biology, oncology, • toxicology, teratology, stem cells • research, regenerative medicine, ... • Regeneration of fins, heart, skin, brain in larval stage – process of rediferentiation, histone demetylation – active proces of rediferentiation, demetylation of histones – "active„ stem cell-like state

  10. chicken embryo (Gallus g. domesticus) • Aristotle (350 BC); later 16.st • molecularanalysisofcancerincludingviral • oncogenesis (P.Rous; 1911), carcinogenesis, tumor xenografting, • tumorangiogenesis and cancer metastasis • - chicken embryo is naturally immunodeficient rabbit (Oryctolagus cuniculus) • advantage(body size), bioreactors, development of new surgical techniques • model of atherosclerosis, spontaneous model • of lipid metabolism, heart hypertrophy • genetical glaucoma • transgenic rabbits

  11. rat (Ratus norvegicus) • bigger than mouse (more cells, greater organs, ...) • neurological model, model of hypertension • Spontaneously hypertensive rat (SHR) • - model of essential (or primary) hypertension • - at 5–6 weeks development of hypertension • between 40 - 50 weeks vascular and cardiac • hypertrophy • hypertension, insulin resistance, insulinemia, • hypercholesterolemia, hypertriglyceridemia

  12. Mouse models of human disease

  13. Mouse (Mus musculus) • gravidity 20 days • one litter 8 pups in average • several litters per year • sexual maturity of females cca after 6 weeks, of males 8w • longevity 2-3 years (1 year of human = 14 days of mouse) • genom cca 2.6x109, haploid chromosomes 20 • financial cost (less than rat or rabbit)

  14. Mouse diseasemodels • Natural mutants SCID (Severe Combined Immunodeficiency) NOD (Non-Obese DM-1) ob/ob (ob = obese, natural mutation of leptin) • Induced diseases physically (surgically altered) - heart failure - liver failure chemically - autoimmune diseases – immunization with autoantigen - chemically induced inflammations cell transfer (implantation of tumor cells) • Transgenic models (knock-out, knock-in, e.g. p53)

  15. Methodologicalapproaches • We are interested in particular disease/process and we want to • know the underlying genes – from phenotype to genotype. • - mutagenesis, organism with particular defect, retrospectiv searching for responsive gene (genetic maping/sequencing) • We are interested in particular gene and we want to know its • role in disease – from genotype to phenotype. • - in which part/cells and when is our GOI (gene of interest) active? • - what will happen, if the gene is knocked down/out, altered? • - what will happen, if the gene is switched on uin diferent part or time than is physiological?

  16. Models with naturally occurring mutations(from phenotype to genotype)

  17. Natural mutation causing autoimmune disease Systemic damage: * NZBxNZW: multiorgan autoimmune disease, defects in T cells and antigen presenting cells * BXSB: lupus-like disease. anti-nuclear Abs and total Ig * MRL lpr/lpr (lympho-proliferation): mutation of Fas gene * MRL gld/gld (generalized lymphoproliferative disease): mutation in Fas-L gene Organ damage: * NOD (non-obese diabetic): DM I. type * C3H/HeJ Bir: colitis

  18. NOD (Non-Obese Diabetic) mice • Model od Diabetes mellitus Type I. (IDDM) - naturally developing autoimmune diabetes - the incidence depends on environmental factors - insulitis in 5th week, diabetes in 16th -30th week - more often found in females (20-100%) than males (1-65%) • Other defects: - defect of the function of Treg-s (CD4+CD25+) - lack and functional defect of NK cells - lack of serum hemolytic complement activity (missing C5) - defect in differentiation and function of antigen presenting cells - impaired production of IL-4

  19. NOD mice – similarity to human DM Type I. • pancreas-specific auto-antibodies • autoreactive CD4+ and CD8+ T lymphocytes • phenotype is polygenically controlled • >20 loci (Idd locus - ”Insulin dependent diabetes”) associated with the disease over at least 11 different chromosomes • Idd1 locus, ch17 essential for disease (symptomatic diabetes) development in NOD mice • encodes the b chain of I-A, an MHC class II protein - Idd4 locus, ch11 • responsible for overexpression of IL-12p40

  20. Models with induced mutations and diseases

  21. Immunization of susceptible strains with „auto-antigen“ • Experimental autoimmune encephalomyelitis (EAE) • Myelin basic protein (MBP) • Proteolipid protein (PLP) • Myelin oligodendrocyte protein (MOG) • Experimental myastenia gravis (EMG) • Torpedo AChR • Experimental arthritis • Colagen typ II – stimulates inflamation • HSP – mildness symptoms • Experimental uveoretinitis • S-antigen (arrestin) • IRBP

  22. Experimental asthma bronchiale (C57BL/6 mice; IL4tm1Cgn): immunization by dendritic cells with ovalbumin (OVA-DC) subsequently exposed to OVA-aerosol no abnormalities, healthy lungs lungs after immunisation with OVA-DC peribronchial and perivascular eosinophil-rich infiltrates „goblet cell “ hyperplasia H&E, 400x Ref: Lambrecht B.N., J Clin Invest, vol. 106, pp. 551-559, 2000.

  23. Experimental insulitisin Balb/c mice:immunization with insulin peptide B:(9-23) with poly-IC (adjuvant) Tetanus toxoid plus PolyIC no insulitis a B:9–23 immunization pankreas no insulitis b B:9–23 plus PolyIC induced insulitis of Langerh. islets d B:9–23 plus PolyIC induced insulitis of Langerh. islets c PNAS 99:5539-5544

  24. Induction by cell transferImmunodeficient strains – model ofcarcinogenesis

  25. Nu/Nu mice (1962; Dr. N.R.Grist) • spontaneous deletion of Foxn1; identified in 1994 • Defect in thymus development: 1. antibody formation 2. cell-mediated immune responses 3. delayed-type hypersensitivity responses 4. killing of virus-infected or malignant cells 5. graft rejection • Model is used for solid cancer studies Before discovery of the nude mouse, human tumors were grafted and grown in immune-privileged sites (anterior chamber of the eye, the brain and the cheek pouch)

  26. SCID (Prkdc/ Scid) mice • 1983 (Blunt et al., PNAS, 1996) • Homozygous mutation of Protein kinase DNA-activated catalytic polypeptide gene, (chr.16) • DNA-dependent protein kinase - activated by DNA double strand breaks - required to generate the antigen binding sites of T-cell receptor and immunoglobulin molecules • Defect in V(D)J recombination - block in differentiation of T and B cells • Defective DNA double-strand break repair - risk of cancer (spontaneous lymphoma)

  27. SCID mice • Similar phenotype to human SCID • >50% of human SCIDs are X-linked • mutations of ADA, IL2R common gamma chain • Cancer studies • engraftment of primary human cancer cells • Hosts for human immune system tissues • HIV studies • Disadvantage „Leaky phenotype“ • Immunoglobulin levels increase with age • short live span (~ 6 month)

  28. Transplantation/xenotransplantation of cells • Transplantation of immunocompetent cells (syngeneic immunodeficient recipient) chronic multiorgan inflammations • Transplantation of syngeneic tumor cells • Xenotransplantation of human tumor cells (NOD/SCID-IL2Rgamma null = NOG) Human & Bovine Intestinal Xenografts in SCID mice to study Crohn’s disease

  29. Transgenic models (from genotype to phenotype)

  30. Transgenic animals • 1974 Rudolf Jaenisch (first transgenic animals - mice) • using the technique of recombinant DNA (does not occur naturally) • the function of GOI (gene of interest) in tissues/diseases - knock-out models (KO) - models with overexpression of one allele - knock-in models (KI) - KO models of regulating or important genes related to GOI - cross-breeds with other models (increasing complexity)

  31. Generating a transgenic animal B) • pronuclear microinjection of DNA into a zygote (generally male) – transgenic mice • Injection of ES cells into blastocysts - chimeric mice • introduction of embryos into pseudopregnant females • production of chimeric mice with mutation or deletion heterozygous mice (+/-) (+/Δ) with mutation or deletion homozygous mice (-/-) (Δ/Δ) with mutation or deletion

  32. (Rosenthal N and Brown S, 2007)

  33. Creation of conditional knock-out preparation of the construct (in vitro) electroporation of ES cells (e.g. From the strain 129S2– donor strain – „agouti“ - brown) homologous recombination in ES cells selection of ES cells - positive (NEO) neomycin, geneticin - negative (HSV1-TK1) ganciclovir – incorrectly incorporated plasmid verifying by PCR/Southern blot Deletion of NEO cassete heterozygout ES cells injected into blastocysts (e.g. strain C57BL/6J - background strain – “black”) Genotyping of offsprings – tail DNA and verifying by PCR/Southern blot Cross-breeding with Cre recombinant animals to create knock-out allele

  34. Mouse models of Li-Fraumeny syndrome

  35. Li–Fraumeny syndrome (LFsy) • rare autosomal dominant inherited predisposition to cancer associated with mutation in gene TP53 • SBLAsyndrome (sarcoma, breast, leukemia, adrenal glad); brain tumors - glioblastomas, lungs, bone sarcomas and lymphomas • the spectrum of TP53 mutations is similar to somatic mutations by spontaneous tumors • tumor incidence (except skin cancer) is 50% within 30 years and 90% within 70 years, overall 100% in women and 73% in men • 25% higher risk of development of malignat tumor

  36. p53 • transcription factor "the guardian of the genome" • tumor suppressor protein • mutation – the most frequent genetic anomaly in human cancer cells • regulates cell response to cytogenotoxic stress and DNA damage (UV, Irm hypoxia, malnutrition, ...) • induces DNA repair, cell cycle arrest in G1/S/G2 phase, inhibition of angiogenesis, formation of metastasis, apoptosis and cell senescence

  37. Mouse models related to p53 I. • first model of overexpression p53 (A135V) Lavigeur et al. 1989 • models with deletion of p53 Donehower et al. 1992, Jacks et al. 1994 • both models – tumor-prone • p53 is dispensable for normal development of an organism Donehower & Lozano 2009 Cancer

  38. Mouse models related to p53 II. mouse p53m mouse p53 A135V Swap p53 p53 super

  39. Our p53 model Nomenclature:C57BL/6J -Trp53tm1Tyj/J 129S2 – donor strain – “agouti”; C57BL/6J(background strain – “black”) transformation related protein 53; targeted mutation 1 The Jackson Laboratory; Tyj Dr. Tyler Jackset al. 1994 Current Biology p53-/- Survival curves and tumor formation in p53-/- p53+/- a wt Tumor distribution by p53+/- ap53-/- mutants Jacks et al. 1994 Current Biology

  40. Similarity of p53 mouse models to LFsy • - 20% LFsy – non functional allel of p53 – KO mutace • the rest are hot-spot mutations R175H and R273H (in mouse R172H and R270H) • however brest cancer (typical for human LFsy) occures with low frequency • the influence of genetic beckground • C57BL/6 vs. Balb/c • Balb/c p53+/– – 55% of females develop brest cancer, more “LFsy-like”

  41. The importance of animal models in pathophysiology of diseases • the role of genes and epigenetic factors in diseases pathogenesis • identification of genes responsible for modification of clinical symptoms of the disease • degree of environmental influence • elucidation of regulating mechanisms • testing of new therapeutic procedures • development of new diagnostic procedures

  42. Links to animal model suppliers Charles River laboratories: http://www.criver.com The Jackson laboratory: http://www.jax.org/ In Czech Republic: Anlab, Velaz: http://www.anlab.cz; http://www.velaz.cz/

  43. Thank you for your attention karin.vargova@gmail.com

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