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FLEXGene Repository
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Transcript of FLEXGene Repository
FLEXGene Repository
Applications
Exploiting FLEXGene
Genes
FLEXGene Clones
Expression Clones
Proteins
Protein ExpressionLocalizationInteractionFunctional Assay
Protein Microarrays
Antibodies
StructuralGenomics
Drug Screens
FLEXGene Repository: Applications
• Accelerates target identification– Functional assays identify biologics– Functional assays identify new drug targets
• Accelerates target validation– Offers immediate access to protein families
• Sorts post-genomic data• Enables pre-clinical toxicology screening• Access to high-throughput biochemistry
– Allows parallel in vitro assays– Promotes rapid development of high-affinity
reagents– Feeds into structural genomics
• Allows gain-of-function tests in mammals– Identifies regulatory proteins
• Enriches database of all protein features– Collects both positive and negative results
Proof of Principle Applications Using
FLEXGene
• High-throughput protein production and purification
• Isolation of genes by functional analysis in mammalian cells
Rapid Screening of Proteins for Interactions
Protein Micro-arraysDrug/Protein linked to marker
Microscopic array of proteins Find a protein target from among thousands
Or do the same process with more than one chemical at a time
But how can thousands of proteins be produced for such arrays?
30 genes 4 different expression vectors = one day
Western Blot Showing Protein Expression of Pilot Constructs
Automated Purification of His-tagged Proteins
Coomassie-stained SDS PAGE of 10% of final purified protein from 1 ml cultures
High Throughput Protein Purification
GST
His
Proteins expressed in bacteria in 96-well blocks and purified in 2 hrs.Methods work for both denaturing and non-denaturing conditions.
Analyzing many proteins identifies features that predict purification success.
Proof of Principle Applications Using
FLEXGene
• High-throughput protein production and purification
• Isolation of genes by functional analysis in mammalian cells
Mammary Epithelial Oncogenesis
Lamellipodia
Jnk
Akt
Integrin Signaling Breast Cancer Genes
Cluster of acini withinmammary lobular unit
L - lobules D - ducts ETD- Extralobular terminal duct
Functional Unit of Human Breast
Mammary gland ducts and lobular units
L - lobules D - ducts
ETD- Extralobular Terminal Duct
Functional Unit of Human Breast
Nuclei
E-Cadherin
Basement membrane
CSWhole mount
Mammary Acini
-CateninColl IV
Acini with milk
Joan Brugge, et al.
Mammary Epithelial Oncogenesis
Lamellipodia
Jnk
Akt
Integrin Signaling Breast Cancer Genes
Lamellipodia
Jnk
Akt
Integrin Signaling Breast Cancer Genes
Carcinoma in situ
Cluster of acini withinmammary lobular unit
L - lobules D - ducts ETD- Extralobular terminal duct
Functional Unit of Human Breast
Mammary gland ducts and lobular units
Invasive carcinoma
L - lobules D - ducts
ETD- Extralobular Terminal Duct
Functional Unit of Human Breast
Nuclei
E-Cadherin
Basement membrane
CSWhole mount
Mammary Acini
-CateninColl IV
Acini with milk
Joan Brugge, et al.
In Vitro Morphogenesis
Basement Membrane
Nuclei
E-Cadherin
Mammary Acini
Senthil Muthuswamy
b-catenin Collagen IVPhase
Matrix gel
BreastEpithelial Cell
Joan Brugge, et al.
Growthfactors Remodeling
Growthfactors
Breast epithelial cells can now be grown in culture to mimic architecture in vivo.
In Vitro Morphogenesis
Growthfactors
Matrix gel
BreastEpithelial Cell
Joan Brugge, et al.
Remodeling Growthfactors
Invasion Hyperplasia
??
?Can we identify genes whose expression causes these cells to behave like cancer cells?
Master ClonesFrom FLEX
Rapid Transfer
High-ThroughputFunctional Assay
CMVVector
+
Expression Vector
“Expression-Ready” Clones
Two “hits” found already.
Many genes tested in parallel
Which genes induce migration in epithelial cells?
Which genes allow cells* to migrate?
No cells made it through
Cells on this side of the membrane
GFP HGF Raf
*MCF10A Cells