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Instituteof Laboratory Animal Science University of Zuric

Transgenic Mouse Models

Institute of Laboratory Animal Science, University of Zurich


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Instituteof Laboratory Animal Science University of Zurich

Transgenic Animals:Definition

Mutant animals carrying experimentally introducedforeign 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: comprisinghomologies to murine sequences


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Gene Insertion

Random

Targeted

Subject to position effects

Insertion by nuclear DNA

repair/recombinationmechanisms


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The Mouse genome


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Genetic NetworksGenes Phenotype


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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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Why the Mouse?

The closest to humansmammal

The most complex -integration of systems (endocrine, immune, nervous etc.)

Of the model organisms which may be geneticallymodified, the mouse is:

Genetic manipulation is extremely versatileGain-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 rolea gene plays in a biological process at the level of thewhole 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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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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Transgene DNA

Analyse the progenyby PCR or Southern blotting

Production of transgenic miceby pronuclear microinjection


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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 intothe host genome and that it is heritable in at least one of the offspring

Demonstrating that the gene is expressed and regulated correctly inthe host organism


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Microinjection station


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Production of transgenic mice

by ES cell gene transfer


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C57BL/6Blastocyst

(black)

ES cells129/SvJ(agouti)

agouti black

ES cell injection into blastocyst


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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 LVinjection

gestation

maturationof founders

Timeline: Transgenesis by PronuclearMicroinjection or Lentiviral transfection

gestation

Birth

Identyfyfounders

maturationof F1 progeny

Beginanalysis


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Timeline: generation of ES cell-derived mice

Introducetargetingvector intoES cells

Identifyhomologousrecombinantsby DNAanalysis

Identify mouseChimeras withhigh ES cellcontribution Germline transmission

Beginanalysis

0 2 4 8 10 126

Drugselection Colony growthand expansion Injectclonesintoblastocysts

Sexualmaturationof chimeras

Identifymale andfemaleheterozygotes

Sexualmaturation ofheterozygotes

Identifyhomozygotes

gestation gestation


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Trends in the Field of Transgenic Animals

More refined transgene systems:

- temporal regulation (tet ON/OFF)

- tissue specific regulation ( Cre/lox)

- RNAi induced gene silencing- YAC/BAC transgenesis

- dominant negative transgenes

IntegrativeDatabases Animal Welfare Aspects


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Transgenic Animals: Potential

Problems

Technical problems to closely mimic adesired situation

Underestimation of biological complexity

Mouse Human differences

Inappropriate analysis

Undefined genetic backgrounds


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Paper to Read

Brinster R.L. et al.: Factors affecting the

efficiency of introducing foreign DNA intomice by microinjecting eggs. Proc. Natl.Acad. Sci USA 82, 4438-4442 (1985).

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