FLEXGene Repository

1. FLEXGene Repository Applications

2. Exploiting FLEXGene Genes FLEXGene Clones Expression Clones Protein Expression Localization Interaction Functional Assay Proteins Drug Screens Structural Genomics Protein Microarrays Antibodies

3. 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

4. Proof of Principle Applications Using FLEXGene • High-throughput protein production and purification • Isolation of genes by functional analysis in mammalian cells

5. 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?

6. Western Blot Showing Protein Expression of Pilot Constructs 30 genes  4 different expression vectors = one day

7. Automated Purification of His-tagged Proteins Coomassie-stained SDS PAGE of 10% of final purified protein from 1 ml cultures

8. High Throughput Protein Purification His GST Proteins expressed in bacteria in 96-well blocks and purified in 2 hrs. Methods work for both denaturing and non-denaturing conditions.

9. Analyzing many proteins identifies features that predict purification success.

10. Proof of Principle Applications Using FLEXGene • High-throughput protein production and purification • Isolation of genes by functional analysis in mammalian cells

11. Mammary Epithelial Oncogenesis Acini with milk Mammary gland ducts and lobular units Cluster of acini within mammary lobular unit Joan Brugge, et al.

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

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

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

15. Which genes induce migration in epithelial cells? Master Clones From FLEX High-Throughput Functional Assay “Expression-Ready” Clones Two “hits” found already. Rapid Transfer + CMV Vector Expression Vector Many genes tested in parallel

16. Which genes allow cells* to migrate? GFP HGF Raf No cells made it through Cells on this side of the membrane *MCF10A Cells