1. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA SMBCa tools

2. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA ENTRY-Clone attL1attL2 gene KAN R attB1attB2 gene AMP R attR2 attR1 AMP R attL1attL2 KAN R attR1attR2 neg. selection AMP R Expressionvector + SMBCa tools : Gateway™ recombination system

3. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA attL1attL2 attR3attR4 ENTRY-Clones Destination Vector attL1attL2 gene KAN R Promoter KAN R attL2RattL3 shRNA against UTR KAN R AMP R X X X attL4attL1R attL4attL1R attL2R attB4 Expression Vector AMP R attB1attB2 attB3 attL3 SMBCa tools : Gateway™ multisite recombination

4. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA ENTRY-Clone attL1attL2 gene KAN R attR1attR2 neg. selection AMP R Expressionvector ~ 16,200 human fully sequenced genes (http://www.orfeomecollaboration.org)http://www.orfeomecollaboration.org reporter constructs for 40 cellular signalling pathways  miRNAs and shRNA  mutation constructs  promoter regions  TF-bindings sites  fusion-tags ~ 16,200 human fully sequenced genes as lentiviral expression constructs expression vectors for native protein expression or protein fusions (fluorescent-, immunogenic- or purification tags) retroviral and lentiviral expressions systems inducible expression vectors SMBCa tools : Entry-clone resources

5. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA RTK P P siRNA / drugs / compounds expression / activation of pathway specific Transcription Factors (TF) binding of TFs to specific consensus sequences on reporter construct  TF binding sites (pathway specific ) reportergene (Firefly Luciferase / RFP) Read – out signal SMBCa tools : Cell-based pathway activity assays

6. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA Cell line (normal / tumor) Acceptor cell line Expression cell line Stable integration of :  Recombination site (FRT)  Resistance gene (Neo)  EGFP Marker Recombination of :  Inducible expression constructs (genes / shRNA/ miRNAs)  Resistance gene (Hyg) Stable isogenic recombinant (Sir) cell lines

7. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA Acceptor cell lines available Skin : A375 (malignant melanoma) Lung : A549 (carcinoma) Lung : H1299 (carcinoma; non-small cell lung cancer) Brain : U373 (alioblastom astrocytom) Breast mammary gland : MCF-7 (adenocarcinoma) MDA-MB231 (adenocarcinoma) Cervix : HeLa S3 (adenocarcinoma)

8. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA Stable isogenic recombinant (Sir) cell lines -Advantages - Transfection independent  low variations Integration of expression constructs in a defined genomic site Single integration of expression constructs Inducible expression  low toxicity Long term expression  drug testing Easy to scale up

9. 3/30/2015 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA Stable isogenic recombinant (Sir) cell lines - Downstream applications - Xenografts + in vivo imaging Protein Quantification / Pathway Identification Echeverri & Perrimon Nature Reviews Genetics 7, 373-384 (May 2006) Drug siRNA/miRNACompount / miRNA Screening Profiling Protein (Silac)RNA / miRNA / CpGs Co-culture / tumor+ stroma cells Cell-based pathway activity assays