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Transgenic Mouse Models Bio 426 / HS 2012. Kurt Bürki, Pawel Pelczar 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 - PowerPoint PPT Presentation
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Institute of Laboratory Animal Science University of Zurich
Transgenic Mouse ModelsBio 426 / HS 2012
Kurt Bürki,
Pawel PelczarInstitute 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
Institute of Laboratory Animal Science University of Zurich
Transgenic Animals: Definition
Mutant animals carrying experimentally introduced foreign genetic elements in all their cells, including
the germline
Institute of Laboratory Animal Science University of Zurich
Steps towards a Transgenic Model
• Working hypothesis
• Gene Construct
• Insertion into an early embryonic stage
• Screening for transgenic animals
• Profiling of expression pattern
• Phenotyping
• Model Validation / Experimentation
Institute of Laboratory Animal Science University of Zurich
Gene Construct
• Expression constructs (transgenes)
• Viral vectors: retroviral/lentiviral vectors
• Targeting constructs: comprising homologies to murine sequences
Institute of Laboratory Animal Science 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)
The Mouse genome
Institute of Laboratory Animal Science University of Zurich
Genetic NetworksGenes Phenotype
Institute of Laboratory Animal Science University of Zurich
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
• Loci, Genes
• Position Effects
• Variegated Expression
• Penetrance
• Expressivity
• Polygenic Traits
• Genetic Background
• Phenotype
Institute of Laboratory Animal Science University of Zurich
GenomeMammalian20 chromosomes2.6 Gb~25000 genes99% have human counterpart
StrainsInbredOutbredRecombinant inbredConsomicFluorescent
Life Cycle4-day oestrus20-day gestation4-8 pups per litter2-8 litters per female7 weeks to sexual maturity2-3 year lifespan
Reverse geneticsKnockoutsTransfenicsConditional expressionInducible expressionRetroviral vectorssiRNA
The Mouse as an Experimental System
Assisted reproductionCryopreservationEmbryo rederivationIn vitro fertilizationIntracytoplasmic sperm injectionCloning
ToolsGenome sequenceEmbryonic stem cellsExpression arraysGene-trap libraries Insertional vector librariesBAC libraries
Institute of Laboratory Animal Science University of Zurich
Why the Mouse?
• The closest to humans – mammal
• The most complex - integration of systems (endocrine, immune, nervous etc.)
Of the model organisms which may be genetically modified, the mouse is:
• 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- 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
Institute of Laboratory Animal Science University of Zurich
Generation of transgenic animals
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Institute of Laboratory Animal Science University of Zurich
Mouse Transgenesis Methods
Pronuclear microinjection
Lentivral infection
ES based transgenesis
pros
Relatively simple and efficientLong transgenes possible
Potentially all species
Very efficientSingle copy insertions
No technical equipmentWorks in many species
Long transgenes possibleGene targeting possibleSingle copy insertions
cons
Random integrationMulticopy insertions( Strain limitations)
High embryo mortality9.5 kb packaging limit
Safety issues (?)Only random integration
Technically difficultTime consuming
Species / Strain limitations
Institute of Laboratory Animal Science University of Zurich
Pronuclear Microinjection
• 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
Institute of Laboratory Animal Science University of Zurich
Microinjection Station
Institute of Laboratory Animal Science University of Zurich
Blastocysts d 3-4 ES-Cell-Colonies
Establishment of ES Cells in vitro
ICM (Innere Zellmasse)
Institute of Laboratory Animal Science University of Zurich
Germline male chimera (C57BL/6 in BALB/c) with offspring
Institute of Laboratory Animal Science University of Zurich
Matefounders
0 2 4 8 10 126
DNA or LV injection
gestationmaturation of founders
Timeline: Transgenesis by Pronuclear Microinjection or Lentiviral transfection
gestation
Birth
Identyfyfounders
maturation of F1 progeny
Beginanalysis
Institute of Laboratory Animal Science University of Zurich
Timeline: generation of ES cell-derived mice
Introducetargeting vector into ES cells
Identifyhomologousrecombinants by DNA analysis
Identify mouse Chimeras with high ES cell contribution Germline transmission
Begin analysis
0 2 4 8 10 126
Drug selection
Colony growth and expansion
Inject clones into blastocysts
Sexual maturation of chimeras
Identifymale and female heterozygotes
Sexual maturation of heterozygotes
Identify homozygotes
gestation gestation
Institute of Laboratory Animal Science University of Zurich
Trends in the Field of Transgenic Animals
• More Refined Transgene Systems:
- temporal regulation (tet ON/OFF)
- tissue specific and temporal regulation ( Cre/lox)
• Gene Targeting in Species other than the Mouse
• Integrative Databases
• 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)
Institute of Laboratory Animal Science 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 Site- Directed Genome Modifications
Institute of Laboratory Animal Science 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
Institute of Laboratory Animal Science 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).