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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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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The Target The Zygote

• Fertilized Egg Accompanied by Usally One Polar
  Body
• Zona Pellucida (Glycoprotein Layer)
• Maternal (Close to Polar Body) and Paternal
  Pronucleus

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Optimal Conditions for the Generation of
Transgenic Mice via Pronuclear MicroinjectionBrin
ster et al. PNAS 82, 4438-4442 (1985)

• Strain Hybrid
• Site of Injection (Paternal) Pronucleus
• DNA Form Linear ends staggered, similar or
  dissimilar
• DNA Size Any few kb, BACS, YACS
• DNA Concentration 1ng/microliter (few
  hundred molecules per nucleus)
• Buffer 10mM Tris-HCl/0.25mM EDTA, pH 7.5

• Average Efficiency Under Optimal Conditions
• Eggs Injected 100
• Eggs Transferred 80
• Pups Born 20
• Transgenic Founders 3

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Pronuclear Microinjection - Advantages

• Realtively simple and efficient
• Coinjection of transgenes possible
• Long transgenes possible
• Potentially all species
• Dominant effects (when transgene is expressed)

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Pronuclear Microinjection - Disadvantages

• Random insertion
• Multiple (Tandem) copies inserted
• Several to many individual lines may have to be
  tested for usefulness
• Strain limitations

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Consequences of Random Insertion

• Position effects influence transgene expression
• Insertion into endogenous genes lead to
  Insertional Mutations (estimated 10 of the
  founders)

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Position Effects

• Denotes the different expression of a given gene
  when trans-located in the genome to different
  neighbouring chromatin.
• Position effects are taken as explanations for
  the often highly different expression levels and
  patterns of randomly inserted transgenes.

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Penetrance

• Denotes the failure of some animals with a mutant
  genotype to express the associated mutant
  phenotype. In any case where less than 100 of
  genotypically mutant animals are phenotypically
  mutant, the phenotype is said to be incompletely
  penetrant. Incomplete penetrance is usually a
  matter of chance or modifiers in the genetic
  background.

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Expressivity

• Denotes the observed quantitative difference in
  the expression of a phenotype among individuals
  that have the same mutant genotype. When
  quantitative differences are observed, a
  phenotype is said to show variable
  expressivity, which can be caused by
  environmental factors, modifier genes or chance.

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Penetrance/Expressivity
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Factors Influencing Transgene Expression/Penetranc
e

• Cis acting Factors
• Regulatory Elements (Promoter, Enhancers, Locus
  Control Regions)
• Copy Number
• Vector Sequences (have negative effects)
• Integration Site (Position Effects)
• Epistatic Effects of Recombined Sequences (eg.
  Metallothionenein Regulatory Seq. Growth
  Hormone Coding Sequences)

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Factors Influencing Transgene Expression/Penetranc
e

• Trans acting Factors
• Epigenetic Modifications (Methylation)
• Imprinting (Germ-line dependent methylation)
• Modifier Genes (Genetic Background)
• DNA Binding Proteins

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In vitro Functional Testing of Transgenes

• The in vitro functional testing of transgenes is
  unreliable (transgenes usually are expressed)
• The ultimate test is the in vivo test

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Promoter-driven Transgenes
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Enhancer-driven Transgenes
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hGH mRNA Expression
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Minilocus Locus Control Region
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Transgene Expressing cDNA
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hAPP Transgenes
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mRNA Expression
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Expression at the Cellular Level
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Variegated Gene Expression

• Differential (heterocellular) gene expression in
  given cell type of an individual
• Characteristic of many transgenes

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Refinements when Designing Transgenes

• BACs, (YACs)
• Targeted Transgenesis (in frame insertion of
  promoterless transgene sequences in predetermined
  locus expression pattern (and level) determined
  by target gene)

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The Inheritance of Transgenes

• Founders Usually in a Mendelian Ratio (50)
• Exceptions Frequent (lt 50 Mosaics
• gt 50 Multiple Integration Sites)
• Offspring Mendelian Ratio (50)

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Paper to Read

• Kistner et al. Doxycycline-mediated quantitative
  and tissue-specific control of gene expression in
  transgenic mice. Proc. Natl. Acad. Sci. USA 93,
  10933-10938 (1996).