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SMBCa tools

SMBCa tools. ENTRY-Clone. Expressionvector. KAN R. AMP R. +. attL1. attL2. attR1. attR2. gene. neg. selection. KAN R. attL1. attL2. attR2. attR1. AMP R. AMP R. attB1. attB2. gene. SMBCa tools : Gateway ™ recombination system. KAN R. KAN R. KAN R. ENTRY-Clones. attL1.

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SMBCa tools

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  1. SMBCa tools

  2. ENTRY-Clone Expressionvector KANR AMPR + attL1 attL2 attR1 attR2 gene neg. selection KANR attL1 attL2 attR2 attR1 AMPR AMPR attB1 attB2 gene SMBCatools : Gateway™ recombination system

  3. KANR KANR KANR ENTRY-Clones attL1 attL2 attL4 attL2R attL3 gene Promoter shRNA against UTR attL1R attL1R attL2R attL3 X attL1 X attL4 X attR3 attB3 Destination Vector attL2 attB2 AMPR attR4 attB1 Expression Vector AMPR attB4 SMBCatools : Gateway™ multisite recombination

  4. ENTRY-Clone Expressionvector KANR AMPR attL1 attL2 attR1 attR2 gene neg. selection SMBCatools : Entry-clone resources • ~ 16,200 human fully sequenced genes (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

  5. siRNA / drugs / compounds P expression / activation of pathway specific Transcription Factors (TF) P SMBCatools : Cell-based pathway activity assays RTK binding of TFs to specific consensus sequences on reporter construct  TF binding sites (pathway specific ) reportergene (Firefly Luciferase / RFP) Read – out signal

  6. Stable isogenic recombinant (Sir) cell lines Cell line (normal / tumor) Stable integration of : • Recombination site (FRT) • Resistance gene (Neo) • EGFP Marker Acceptor cell line Recombination of : • Inducible expression constructs (genes / shRNA/ miRNAs) • Resistance gene (Hyg) Expression cell line

  7. Breast mammary gland : MCF-7 (adenocarcinoma) MDA-MB231 (adenocarcinoma) Brain : U373 (alioblastom astrocytom) Cervix : HeLa S3 (adenocarcinoma) Lung : A549 (carcinoma) Lung : H1299 (carcinoma; non-small cell lung cancer) Skin : A375 (malignant melanoma) Acceptor cell lines available

  8. 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 Stable isogenic recombinant (Sir) cell lines -Advantages -

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

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