Transgenic Animals, Knock-Out Mice and Dolly

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Pluripotent ES Cells

• Pluripotent ES cells are undifferentiated early
  embryonic cells derived from the inner cell mass
  of mouse blastocysts.
• In vitro ES cells must be grown on a feeder layer
  of fibroblasts to prevent them from
  differentiating.
• Introduction of the transgene is achieved by
  electroporation of retroviral infection.
• The transgene must integrate via recombination,
  not randomly.
• Cells transfected successfully can be identified
  prior to injection into blastocysts.

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Specific Gene Targeting in ES Cells

• Gene targeting can be achieved using gene
  constructs designed for homologous recombination.
  This technique can be used to either
• Knockout functional genes to study their
  contribution to different developmental or
  disease processes (null mutations)
• Genes encoding b2m, MHC class I and II. CD2, Ii,
  TCR, Ig, IL-4, IL-2, FceR, TAP1/2, RAG-2,and many
  more (gt100)!
• Replace a functional gene for a
  mutated/non-functional gene to restore wild type
  phenotype .
• Gene encoding HGPRT in mice deficient for HGPRT
  (called Lesch-Nhyan syndrome in humans).

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DNA Constructs for Recombination

• DNA vectors contain the gene of interest which
  has been interrupted with an antibiotic
  resistance gene (hygromycin resistance, or G418
  resistance).
• To ensure targeted integration has occurred, the
  flanking DNA contains the thymidine kinase gene.
  If TK integrates (random insertion), then the
  transfected cells die when grown in selective
  media (gancyclovir).

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Selection of Targeted ES Cells

• Gancyclovir resistant and G418 resistant ES cells
  grow into small clumps on top of feeder cells.
• The colonies of cells can be picked off and
  transferred to new wells (at 0.3 cells per well
  seeding density) containing feeder cells.
• When sufficient numbers of cells are obtained,
  they are
• Frozen for safe storage
• Analyzed by Southern blotting or PCR to determine
  nature of integration event
• Microinjected into the blastocoel cavity of
  blastocysts.

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• http//www.criver.com/products/genetic_testing/dxg
  mon2.html

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Dolly and the Advancement of Animal Cloning
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