FLEXGene Repository Applications

Exploiting FLEXGene Genes FLEXGene Clones Expression Clones Proteins Protein Expression Localization Interaction Functional Assay Protein Microarrays Antibodies Structural Genomics 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-arrays Drug/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 Cluster of acini within mammary lobular unit Mammary gland ducts and lobular units Acini with milk Joan Brugge, et al.

Mammary Epithelial Oncogenesis Carcinoma in situ Cluster of acini within mammary lobular unit Mammary gland ducts and lobular units Invasive carcinoma Acini with milk Joan Brugge, et al.

In Vitro Morphogenesis b-catenin Collagen IV Phase Matrix gel Breast Epithelial Cell Joan Brugge, et al. Growth factors Remodeling Growth factors Breast epithelial cells can now be grown in culture to mimic architecture in vivo.

In Vitro Morphogenesis Growth factors Matrix gel Breast Epithelial Cell Joan Brugge, et al. Remodeling Growth factors InvasionHyperplasia ? ? ? Can we identify genes whose expression causes these cells to behave like cancer cells?

Master Clones From FLEX Rapid Transfer High-Throughput Functional Assay CMV Vector + 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 GFPHGFRaf *MCF10A Cells