18. Transgenic Models

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18. Transgenic Models
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Approaches Used in the Analysis of Mammalian
Development

• Observations during embryogenesis
• Phenotypic analysis of developmental mutants
• Cloning and analysis of expression of
  developmental genes
• Generation of mouse mutants by gene targeting
• Gene transfer in established cell lines and
  transgenic mice
• Nuclear transfer (cloning)

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Transgenesis definitions

• Transgenic animal one that carries a foreign
  gene that has been deliberately inserted into its
  genome.
• Chimeric animal one that carries an altered
  gene introduced using manipulated embryonic stem
  (ES) cells. Some tissues are derived from cells
  of the recipient blastocyst other tissues are
  derived from the injected ES cells.
• Knockout mutation replacement of a gene segment
  by homologous recombination that normally results
  in a nonfunctional or null allele.

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Transgenesis Definitions (continued)

• Knock-in mutation similar to a knockout
  mutation, except mutation is usually a point
  mutation that results in a partially functional
  or nonfunctional allele.
• Nuclear transfer technique used to create a
  clone. The nucleus from an adult somatic
  (differentiated) cell is inserted in the emptied
  cytoplasm of an egg cell. The egg cell
  reprograms the adult cells genes so that it
  behaves like an all-purpose stem cell.

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Organisms utilized as transgenic models

• Arabadopsis (plant)
• C. elegans (worm)
• Fruit flies
• Xenopus (frog)
• Zebrafish
• Mice

• Rats
• Pigs
• Sheep
• Goats
• Cows

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Types of transgenes

• Small recombinant DNA molecules genes or cDNAs
  linked to DNA sequences that allow correct
  expression by the cells of the host
• Reporter constructs desired gene promoter
  linked to expression cassette that can be
  assayed e.g., GFP, lacZ, luciferase
• Large native DNA molecules yeast artificial
  chromosomes (YACs) or bacterial artificial
  chromosomes (BACs)

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• Frogs were among the first transgenic animals
  produced.
• Isolated DNA was not transferred, nuclei were
  utilized (setting the stage for recent cloning
  technology).
• Microinjection methodology was established.

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Mouse Models of Human Disease Utility

• Physiologically similar to humans.
• Large genetic reservoir of potential models has
  been generated through identification of 1000
  spontaneous, radiation- or chemically-induced
  mutant loci.
• Recent technological advances have dramatically
  increased our ability to create mouse models of
  human disease.
• 1. Development of high resolution genetic and
  physical linkage maps of the mouse genome
  facilitates identification and cloning of mouse
  disease loci.
• 2. Transgenic technologies that allow one to
  ectopically express or make germline mutations in
  virtually any gene in the mouse genome i.e.,
  transgenic mice, ES cell knockouts.
• 3. Methods for analyzing complex genetic
  diseases.

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Mouse Models of Human Disease Utility
(Continued)

• 100 mouse models of human disease where the
  homologous gene has been shown to be mutated in
  both human and mouse.
• 1. Mouse mutant phenotype very closely
  resembles the human disease phenotypes.
• 2. Provide valuable resources to understand
  how the diseases develop and test ways to prevent
  or treat these diseases.
• Allow study of disease on uniform genetic
  background.
• Will aid in identifying modifier genes and are
  well poised to lead us into the new era of
  polygenic disease research.

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Production of transgenic mice (overview)
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Production of transgenic mice
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Example of transgenic mouse
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Production of chimeric mice (overview)
Inject genetically modified embryonic stem (ES)
cells from brown mouse into black mouse
blastocyst (embryo)
Implant into foster mother
Brown (or mixed) coat color mice are chimeras
Look for chimeric progeny (coat color)
brown
black
Breed for germline transmission
black
brown
Check offspring for coat color
brown
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Isolation of ES cells
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Gene targeting method to produce null mutant
(knockout) or mutant (knock-in) mice
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Homologous recombination
neo

G418R GANCR
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Random integration
neo
HSV tk
G418R GANCS
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Generation of mutant (chimeric) mouse strain
/ ES cells from brown mouse
electroporate targeting vector
homologous recombination
/- ES cells
microinjection into black embryo
X
chimera
screen chimera progeny
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Chimeras
Non-chimeras are black.
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Production of chimeric mice
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Knockout mice

• Valuable for discovering function(s) of genes for
  which mutant strains were not previously
  available.
• Generalizations
• 1. Mice are often surprisingly unaffected by
  their deficiency. Many genes turn out not to be
  indispensable.
• 2. Most genes are pleiotropic that is, they
  are expressed in different tissues in different
  ways and at different times in development.

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Studies Using Transgenics

• Rescue of mutants
• Mice as bioreactors synthesis of human
  monoclonal antibodies.
• Structure-function relationships
• Mouse models of human disease analysis of
  cellular and molecular mechanisms underlying
  pathogenesis and for testing drug regimes and/or
  gene therapies.

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Mouse models listed by primary organ or tissue
system affected

• Disorders of neural crest derivatives 11
• Disorders of vision and hearing 7
• Disorders of bone, skin and connective tissue
  16
• Neurological and neuromuscular disorders 22
• Neoplastic disorders 11
• Immunological and hematological diseases 17
• Metabolic and hormonal diseases 24
• Human diseases with polygenic etiology 6

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Examples of mouse models of genetic diseases

• Sickle cell disease combination of knockout and
  transgenic technologies. Endogenous globin genes
  made null, human globin genes introduced as
  transgenes.
• Cystic fibrosis mainly knockout models, some
  transgenic.
• Polycystic kidney disease knockout models only.
• Narcolepsy knockout model.

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Narcoleptic mouse, a.k.a. model of medical
students attending early morning lecture.