Transcript Transgenic Mouse Models - Institut für Labortierkunde

Transgenic Mouse Models
Bio 426 / HS 2012
Kurt Bürki,
Pawel Pelczar
Institute of Laboratory Animal Science, University of Zurich
Institute of Laboratory Animal Science
University of Zurich
Goals
• To cover the techniques to generate
transgenic models
• To compare advantages / disadvantages /
limits of the techniques
• To discuss important models in several
fields of biomedical research
Institute of Laboratory Animal Science
University of Zurich
Learning Objectives
By the end of the lecture series the participants are
able to:
• List advantages and disadvantages of the major
methods to generate transgenic animals
• Design functional transgenes and targeting
vectors
• Present and critically discuss original papers in
the field in a comprehensive form (key skill)
Institute of Laboratory Animal Science
University of Zurich
Additional Practical Courses
• BIO 413: Generation of Transgenic Animals (LTK
Module 3E)
• BIO 412: Einführung in die Labortierkunde / Introduction into
Laboratory Animal Science (LTK Modul 1)
Institute of Laboratory Animal Science
University of Zurich
Structure of the Lecture Series
• Introduction
• Technical Aspects (Students: Paper to read /
Comrehensive presentation)
• Transgenic Mouse Models (Students: Paper or
review to read / Identification of questions
relevant for a given field)
• Exam
• Visit of a Laboratory
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University of Zurich
Transgenic Animals: Definition
Mutant animals carrying experimentally introduced
foreign genetic elements in all their cells, including
the germline
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University of Zurich
Steps towards a Transgenic Model
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Working hypothesis
Gene Construct
Insertion into an early embryonic stage
Screening for transgenic animals
Profiling of expression pattern
Phenotyping
Model Validation / Experimentation
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University of Zurich
Gene Construct
• Expression constructs (transgenes)
• Viral vectors: retroviral/lentiviral vectors
• Targeting constructs: comprising
homologies to murine sequences
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University of Zurich
Gene Insertion
• Insertion by nuclear DNA
repair / recombination
mechanisms
• Random (non-homologous
end joining NHEJ: subject to
position effects)
• Targeted (homologous
recombination)
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University of Zurich
The Mouse genome
Genetic Networks
Genes
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Phenotype
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Institute of Laboratory Animal Science
University of Zurich
Institute of Laboratory Animal Science
University of Zurich
Transgenics vs. Genetics
• Transgene
• Promoter/Coding
Sequence
• Insertion Site
• Targeting Vector
• Knock-out/Knock-in
• Conditional Mutants
• Phenotype
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Loci, Genes
Position Effects
Variegated Expression
Penetrance
Expressivity
Polygenic Traits
Genetic Background
Phenotype
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The Mouse as an Experimental System
Genome
Mammalian
20 chromosomes
2.6 Gb
~25000 genes
99% have human counterpart
Life Cycle
4-day oestrus
20-day gestation
4-8 pups per litter
2-8 litters per female
7 weeks to sexual maturity
2-3 year lifespan
Strains
Inbred
Outbred
Recombinant inbred
Consomic
Fluorescent
Assisted reproduction
Cryopreservation
Embryo rederivation
In vitro fertilization
Intracytoplasmic sperm injection
Cloning
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Reverse genetics
Knockouts
Transfenics
Conditional expression
Inducible expression
Retroviral vectors
siRNA
Tools
Genome sequence
Embryonic stem cells
Expression arrays
Gene-trap libraries
Insertional vector libraries
BAC libraries
University of Zurich
Why the Mouse?
Of the model organisms which may be genetically
modified, the mouse is:
• The closest to humans –
mammal
• The most complex integration of systems (endocrine, immune, nervous etc.)
• Genetic manipulation is extremely versatile –
Gain-of-Function (Transgenesis), Loss-of-Function
(knock-out), Change-of-Function (knock-in);
temporally and spatially restricted (conditional)
Institute of Laboratory Animal Science
University of Zurich
Applications of transgenic mice
Transgenic mice are often generated to address the role
a gene plays in a biological process at the level of the
whole organism:
- To confirm the role of a gene mutation
- To help unravel the molecular
mechanisms that control gene expression
- To help unravel the biochemical in vivo mechanisms
and the origin of disease
- To develop an animal model to test therapeutic
strategies
Institute of Laboratory Animal Science
University of Zurich
Transgenic Animals: Methods
Classical
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Pronuclear Microinjection
Lentiviral Infection
Embryonic Stem (ES) Cell Gene Transfer
ES Cell mediated Gene Targeting (knock-out, knock-in)
Experimental
- Transfection of Somatic Cells - Cloning
- Sperm Based Transfection (ICSI)
- Transposons
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University of Zurich
Generation of transgenic animals
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Institute of Laboratory Animal Science
Zur Anzeige w ird der QuickTime™
Dekompressor „TIFF (Unkomprimiert)“
benötigt.
University of Zurich
Mouse Transgenesis Methods
pros
cons
Relatively simple and efficient
Long transgenes possible
Potentially all species
Random integration
Multicopy insertions
( Strain limitations)
Very efficient
Single copy insertions
No technical equipment
Works in many species
High embryo mortality
9.5 kb packaging limit
Safety issues (?)
Only random integration
Long transgenes possible
Gene targeting possible
Single copy insertions
Technically difficult
Time consuming
Species / Strain limitations
Pronuclear microinjection
Lentivral infection
ES based transgenesis
Institute of Laboratory Animal Science
University of Zurich
Pronuclear Microinjection
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Microinjection of DNA directly into the pronuclei of fertilized eggs
Implantation of the microinjected eggs into a surrogate mother
Allowing the embryos to develop to birth
Demonstrating that the foreign gene has been stably incorporated into
the host genome and that it is heritable in at least one of the offspring
• Demonstrating that the gene is expressed and regulated correctly in
the host organism
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University of Zurich
Microinjection Station
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University of Zurich
Establishment of ES Cells in
vitro
Blastocysts d 3-4
ES-Cell-Colonies
ICM (Innere Zellmasse)
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University of Zurich
Germline male chimera (C57BL/6 in BALB/c)
with offspring
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University of Zurich
Timeline: Transgenesis by Pronuclear
Microinjection or Lentiviral transfection
DNA or LV
injection
Identyfy Mate
founders founders
Begin
analysis
Birth
0
gestation
2
maturation
of founders
4
gestation
6
8
10
maturation
of F1 progeny
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University of Zurich
12
Timeline: generation of ES cell-derived mice
Introduce
targeting
vector into
ES cells
0
Drug
selection
Identify
homologous
recombinants
by DNA
analysis
2
Colony growth
and expansion
Identify mouse
Chimeras with
high ES cell
contribution
4
6
gestation
Inject
Sexual
clones
maturation
into
of chimeras
blastocysts
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Germline transmission
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Begin
analysis
10
gestation
Identify
Sexual
male and
maturation of
female
heterozygotes
heterozygotes
12
Identify
homozygotes
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Trends in the Field of Transgenic Animals
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More Refined Transgene Systems:
- temporal regulation (tet ON/OFF)
- tissue specific and temporal regulation ( Cre/lox)
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Gene Targeting in Species other than the Mouse
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Integrative Databases
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Animal Welfare Aspects
Institute of Laboratory Animal Science
University of Zurich
Trends with Transgenic Animals
(1)
Targeted Modifications, Control over Expression or
Silencing, Combined (binary) Systems
• Inducible Transgene-Expression
Tet-on, Tet-off Systems)
• Tissue-specific knock-outs (Cre-lox System)
• Inducible knock-outs (CreMER System)
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University of Zurich
Trends with Transgenic Animals
(2)
• Routine Gene-Targeting in Mammalian
Species other than the Mouse
• New: Gene Targeting in Rat ES Cells / iPS
Cells / Spermatogonial Stem Cells
• New: Zinc-Finger Nucleases
for the Introduction of SiteDirected Genome
Modifications
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University of Zurich
Transgenic Animals: Potential
Problems
• Technical problems to closely mimic a
desired situation
• Underestimation of biological complexity
• Mouse – Human differences
• Inappropriate analysis
• Undefined genetic backgrounds
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University of Zurich
Example: The App Gene
(Alzheimers Disease)
Institute of Laboratory Animal Science
University of Zurich
Paper to Read
Brinster R.L. et al.: Factors affecting the
efficiency of introducing foreign DNA into
mice by microinjecting eggs. Proc. Natl.
Acad. Sci USA 82, 4438-4442 (1985).
Institute of Laboratory Animal Science
University of Zurich