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)

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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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Transgenic Animals: Definition

Mutant animals carrying experimentally introduced foreign genetic elements in all their cells, including

the germline

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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

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Gene Construct

• Expression constructs (transgenes)

• Viral vectors: retroviral/lentiviral vectors

• Targeting constructs: comprising homologies to murine sequences

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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

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Genetic NetworksGenes 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

• Loci, Genes

• Position Effects

• Variegated Expression

• Penetrance

• Expressivity

• Polygenic Traits

• Genetic Background

• Phenotype

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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

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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)

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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

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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

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Generation of transgenic animals

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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

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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

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Microinjection Station

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Blastocysts d 3-4 ES-Cell-Colonies

Establishment of ES Cells in vitro

ICM (Innere Zellmasse)

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Germline male chimera (C57BL/6 in BALB/c) with offspring

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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

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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

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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

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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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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

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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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Example: The App Gene (Alzheimers Disease)

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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).