Jeff Green, M'D'

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Building Models of Disease the revolution of
designer mice
Jeff Green, M.D. Transgenic Oncogenesis
Group Laboratory of Cell Regulation and
Carcinogenesis
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GH mice
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Outline of Presentation

• Overview of technology
• Advantages/Disadvantages
• Applications
• Model Validation

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molecular biology
cell biology
animal models
clinical application
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Genetically Engineered Mice

• Limited only by our imagination!

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Engineer Mice to

• Develop cancer
• Prevent cancer
• Modulate cancer
• Modify specific pathways
• Express and/or secrete proteins (pharmacology)
• Recapitulate specific genomic changes
• Test environmental influences
• Test therapeutic stratagies

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Advantages of GEM Over In vitro Approaches

• Study gene in context of entire animal
• Effects on animal development
• Study cancer stage progression
• Study tissue-tissue interactions
• Study genetic interactions
• Identify modifier loci
• Tools for pre-clinical testing

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Disadvantages

• a mouse is only a mouse!

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Process? Cell Origin?
Genetic Aberration(s)
Epigenetic Changes
Stroma
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Genetic Aberration(s) (All cells)
- p53 - BRCA1
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Designing GEM Modelsa wealth of technologies

• Classic transgenic approach
• Classic knock-out
• Crossing transgenic models

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Transgenic Targeting to the Mammary and Prostate
Glands

• tissue-specificity

• cell-compartment specificity

• developmental timing

• hormone-independent promoter

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STRATEGIES FOR HOMOLOGOUS RECOMBINATION
TK
NEO
II
III
II
III
Insertion
NEO
II
III
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TK
NEO
II
III
II
III
Deletion
NEO
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KNOCK-IN STRATEGY

NEO
TK
II
III
II
III
Replacement

NEO
II
III
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Additional GEM Technologies

• Viral delivery systems (retrovirus/lentevirus)
• RACS system
• Embryonic reconstitution
• Chimeric mice (VEGF ets K.O.s)
• BAC transgenics
• Recombineering (Regeneron)

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Embryonic U.G. Sinus
Retrovirus ras/myc
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Y.Li
The RCAS-TVA Technology
RCAS - a viral vector derived from ALV-A TVA -
avian receptor for ALV-A
SD
SA
SA
gag
pol
env
LTR
LTR
X
X
gag, pol
env
gene X
TVA
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Gene Delivery with RCAS Vectors
tv-a transgenic mouse
Pr
TV-a

• active oncogenes
• DN TSGs
• inducible genes
• Cre recombinase
• Tet activator
• marker genes

gag
pol
env
LTR
LTR
X
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Conditional Expression Models

• Express a gene with some control on when and
  where

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CONDITONAL KNOCK-0UT (
Cre-lox)
L
NEO
TK
L
II
III
II
III
Replacement
L
NEO
NEO
L
II
III
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L
NEO
NEO
L
II
III
X
Promoter
cre recombinase
II
L
NEO
NEO
III
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C3(1)/Tag Transgenic Model
Human
Mouse
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Overview of Molecular Studies
Proliferation
Apoptosis
Cell-cycle regulators
Bcl-2 family members
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Tumor Progression in Breast Cancer
Shibata, EMBO, 2001
? Bax