Transcript Transgenic models

Biological models of diseases
Karina Vargová, and others…
block seminar
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
• model organisms
• mouse models of human diseases with
- natural mutations
- induced mutations
- transgenic organisms
2
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“
3
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
4
How to choose an appropriate animal model?
1) Appropriate to the condition being investigated
2) 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)
3) Well designed experiments
(are capable of demonstrating the response, appropriate size, length of the
animal, adequate controls and amounts of animals for statistical evaluation)
4) Models are highly specific
(may work in some study, but not in other)
5) Clinical study will validate the model (e.g. testing of new drug)
5
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
6
Some typical model organisms
Invertebrates:roundworm (Caenorhabditis elegans)
fruit fly (Drosophila melanogaster)
Vertebrates: platanna (Xenopus laevis)
zebrafish (Danio rerio)
chicken (Gallus gallus domesticus)
mouse (Mus musculus)
rat (Ratus norvegicus)
rabbit (Oryctolagus cuniculus)
... dog, cat, pig, monkey, primates
7
Roundworm
(Caenorhabditis elegans)
-
developmental biology (1974; Sydney Brenner)
genome completely sequenced – 100x106 bp (1998; 2002)
strictly defined somatic cell number (eutely), transparent
(S.Brenner,
H.R.Horvitz, J.
recently study of molecular genetics of aging
Sulston)
Nobel prices: 2002 - development, programmed cell death
(Physiology
2006 - RNA interference
medicine)
(chemistry) 2008 - GFP (M.Chalfie)
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
(A.Fire, C.C.Mello)
8
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 –
9
"active„ stem cell-like state
chicken embryo (Gallus g. domesticus)
- Aristotle (350 BC); later 16.st
- molecular analysis of cancer including viral
oncogenesis (P.Rous; 1911), carcinogenesis, tumor xenograft
tumor angiogenesis 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
10
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
11
Mouse models of human disease
12
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)
13
Mouse disease models
• 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)
14
Methodological approaches
1. 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)
2. 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?
15
Models with naturally occurring mutations
(from phenotype to genotype)
16
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
17
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
18
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
19
Models with induced mutations and
diseases
20
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
21
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. 22
Experimental insulitis in Balb/c mice:
immunization with insulin peptide B:(9-23) with poly-IC (adjuvant)
a
B:9–23 immunization
pankreas no insulitis
c
B:9–23 plus PolyIC
induced insulitis of Langerh. islets
b
d
Tetanus toxoid plus PolyIC
no insulitis
B:9–23 plus PolyIC
induced insulitis of Langerh. islets
23
PNAS 99:5539-5544
Induction by cell transfer
Immunodeficient strains – model of
carcinogenesis
24
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)
25
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)
26
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)
27
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
28
Transgenic models
(from genotype to phenotype)
29
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)
30
Generating a transgenic animal
B)
A) pronuclear microinjection of DNA into a
zygote (generally male) – transgenic mice
B) 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
31
32
(Rosenthal N and Brown S, 2007)
Creation of conditional knock-out
1. preparation of the construct (in vitro)
2. electroporation of ES cells (e.g. From the
strain 129S2 – donor strain – „agouti“ brown)
3. homologous recombination in ES cells
4. selection of ES cells
- positive (NEO) neomycin, geneticin
- negative (HSV1-TK1) ganciclovir –
incorrectly incorporated plasmid
5. verifying by PCR/Southern blot
6. Deletion of NEO cassete
7. heterozygout ES cells injected into
blastocysts (e.g. strain C57BL/6J background strain – “black”)
8. Genotyping of offsprings – tail DNA and
verifying by PCR/Southern blot
9. Cross-breeding with Cre recombinant
animals to create knock-out allele
33
Mouse models of
Li-Fraumeny syndrome
34
Li–Fraumeny syndrome (LFsy)
• rare autosomal dominant inherited predisposition to cancer
associated with mutation in gene TP53
• SBLA syndrome (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
35
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
36
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
37
Donehower & Lozano 2009 Cancer
Mouse models related to p53 II.
mouse p53m
mouse p53 A135V
p53 super
Swap p53
38
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 Jacks et al. 1994 Current Biology
p53-/-
Survival curves and tumor formation in p53 -/- p53+/- a wt
Tumor distribution by p53+/- a p53-/- mutants
39
Jacks et al. 1994 Current Biology
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”
40
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
41
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/
42
Thank you for your attention
[email protected]
43