Institute of Laboratory Animal Science University of Zurich

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Transgenic Mouse ModelsBio 426 / HS 2007
Kurt Bürki, Institute of Laboratory Animal
Science, University of Zurich
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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

• Random
• Targeted
• Subject to position effects
• Insertion by nuclear DNA repair/recombination
  mechanisms

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The Mouse genome
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Genetic Networks
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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?
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)

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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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Mouse Transgenesis Methods
pros Relatively simple and efficient Long
transgenes possible Potentially all
species Very efficient Single copy
insertions No technical equipment Works in many
species Long transgenes possible Gene
targeting possible Single copy insertions
cons Random integration Multicopy insertions (
Strain limitations) High embryo mortality 9.5
kb packaging limit Safety issues (?) Only random
integration Technically difficult Time
consuming Species / Strain limitations
Pronuclear microinjection Lentivral
infection ES based transgenesis
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Analyse the progeny by PCR or Southern blotting
Transgene DNA
Production of transgenic mice by 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 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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Production of transgenic mice by ES cell gene
transfer
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ES cell injection into blastocyst
C57BL/6 Blastocyst (black)
agouti black
ES cells 129/SvJ (agouti)
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Germline male chimera (C57BL/6 in BALB/c) with
offspring
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Timeline Transgenesis by Pronuclear
Microinjection or Lentiviral transfection
DNA or LV injection
Mate founders
Identyfy founders
Begin analysis
Birth
0
2
4
8
10
12
6
gestation
gestation
maturation of founders
maturation of F1 progeny
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Timeline generation of ES cell-derived mice
Identify homologous recombinants by DNA analysis
Identify mouse Chimeras with high ES cell
contribution
Introduce targeting vector into ES cells
Begin analysis
Germline transmission
0
2
4
8
10
12
6
gestation
gestation
Drug selection
Colony growth and expansion
Inject clones into blastocysts
Sexual maturation of chimeras
Identify male and female heterozygotes
Sexual maturation of heterozygotes
Identify homozygotes
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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
• Integrative Databases
• Animal Welfare Aspects

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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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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).