1. Yeast 2-hybrid database Plasmid database ATCC plasmid distribution Alliance for Cell Signaling Myriad Genetics Inc. American Type Culture Collection

2. Yeast 2-hybrid project has switched to a macrophage focus Years 1 and 2 (to May ‘03) –Baits screened against 3 libraries (all mouse): Primary B cell + WEHI231 B cell line Primary myocyte* NK tag myocyte cell line Year 3 (May ‘03-’04) –New macrophage library prepared Resting RAW cells LPS + IFN  stimulated RAW cells Primary bone marrow-derived macrophages –Year 3 baits are currently being screened against 3 libraries Macrophage B cell Mouse embryo (mixture of several embryonic stages)

3. Current status of Y2H screens New macrophage library ready for use late 2003 74 bait proteins selected for screening this year –46 new proteins –28 proteins previously screened against old library set selected for re-screening –See poster #07 for full bait list Data pending on this years screens Completion of screens from years 1 and 2 has provided a large quantity of new data –May 2003473 interactions –May 20041081 interactions

9. Y2H data files included in download

10. 1081 total interactions

12. Y2H data files included in download

13. All 1081 interactions viewed in cytoscape Other AfCS proteins AfCS proteins in FXM pathway Protein with no AfCS ID (GI# known) Protein with no AfCS ID (novel sequence)

14. Yeast 2-Hybrid summary Website and data format improvements significantly enhance utility of Y2H data We expect the current ‘year 3’ screens to provide valuable additional data, especially for proteins central to macrophage signaling pathways Prevalence of FXM specific proteins in this years bait set should provide a wealth of protein-protein interaction data for this network

15. AfCS Plasmid Database

21. Gateway-based plasmid construction

22. Automatic generation of plasmid sequences and feature information attB1 Gene attB2attB1 Gene attB2 attB1 Gene attB2attB1 Gene attB2 Promoter Amp resistance Parent template: Cloned gene: Construct: Promoter Amp resistance

23. Automatic generation of plasmid sequences and feature information Parent: Cloned gene: Construct: +

24. Construct barcode A05XK099A1NK ID of Parent vectorVector type (X=expression) Gene aliasGene versionGene form (N=no termination codon) Bacterial selection (K=kanamicin)

25. Akt1 cloned genes Gene forms and versions describe characteristics of each clone

26. What have we cloned for cDNA expression? Parent vectors (200+, 50+ to ATCC) Fluorescent protein tagging –All GFP variants Epitope tagging –Flag, HA, Myc, TAP Promoter options –CMV, EF1, viral LTRs Expression platforms –Mammalian, retroviral, lentiviral cDNAs Full-length wild type (900+) –Kinases (550+) –Others (350+) Mutants –Dominant –ve (180) –Constit. Active (6) Domains –PH domains (136) –Subcellular markers (50+)

27. What have we cloned for RNAi? Parent vectors –Pol III cloning vectors (11) –shRNA expression vectors Lentiviral (30+) Retroviral (2) Mammalian (10+) –17 to ATCC (website feature) shRNAs –Genes targeted (70+) –Total shRNAs (200+) –Validated shRNA constructs to ATCC (114) attL1 mH1 or mU6 attL2 attR1attR2FLAPLTRUbi-CWRECD4 Flag HA CD8 LTR shRNA IRES or IRES2 Puro Hyg Zeo Neo

28. 2,700+ Plasmids are now available through the ATCC Internal: 220 parents 5000+ constructs Public: 53 parents 2,709 constructs

29. Acknowledgements Molecular Biology Laboratory –Genie Lee –Qingli Mi –Eileen Fung –Joelle Zavzavadjian –Jamie Liu –Sam Couture –Kavitha Dhandapani –Estelle Wall –Leah Santat Microscopy Laboratory –Weisun Park –Sarah Lim –Grischa Chandy Bioinformatics Laboratory –Stephen Lyon –Andreia Maer –Brian Saunders ATCC –Judy Kantor –Linda Holloway –Jennifer Zinck –Barry Westfall Yeast 2-hybrid –AfCS Bob Sinkovits Gil Sambrano –Myriad Genetics Terrece Pearman Brandi Williams Max Dufford Karen Heichman