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