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Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
SMBCa tools
5/23/2017 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
SMBCa tools :
Gateway™ recombination system
ENTRY-Clone
Expressionvector
KANR
AMPR
+
attL1
attL2
attR1
gene
attR2
neg. selection
KANR
attL1
attL2
attR1
attR2
5/23/2017 |
AMPR
AMPR
attB1
attB2
gene
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
SMBCa tools :
Gateway™ multisite recombination
KANR
ENTRY-Clones
attL1
KANR
attL2
gene
attL4
KANR
attL1R
attL2R
attL3
Promoter
shRNA against UTR
5/23/2017 |
Destination Vector
attL4
X
attR4
attL2R
X
attL2
attL1
X
attL1R
attL3
attR3
AMPR
Expression Vector
attB4
attB1
attB2
AMPR
attB3
SMBCa tools :
Entry-clone resources
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
ENTRY-Clone
KANR
attL1
attL2
gene
~ 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
5/23/2017 |
Expressionvector
AMPR
attR1
~ 16,200 human fully sequenced genes as lentiviral expression
constructs
 expression vectors for native protein expression or protein fusions
attR2
neg. selection
(fluorescent-, immunogenic- or purification tags)
 retroviral and lentiviral expressions systems
 inducible expression vectors
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
SMBCa tools :
Cell-based pathway activity assays
P
P
siRNA / drugs / compounds
5/23/2017 |
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
Dr. Rainer Will
Stable isogenic recombinant (Sir) cell lines
DKFZ Heidelberg
GPCF / CIA
Cell line
(normal / tumor)
Stable integration of :



Recombination site (FRT)
Resistance gene (Neo)
EGFP Marker
5/23/2017 |
Acceptor
cell line
Recombination of :


Expression
cell line
Inducible expression constructs
(genes / shRNA/ miRNAs)
Resistance gene (Hyg)
Dr. Rainer Will
Acceptor cell lines available
DKFZ Heidelberg
GPCF / CIA
Breast mammary gland : MCF-7 (adenocarcinoma)
MDA-MB231 (adenocarcinoma)
Brain : U373 (alioblastom astrocytom)
Cervix : HeLa S3 (adenocarcinoma)
5/23/2017 |
Lung : A549 (carcinoma)
Lung : H1299 (carcinoma; non-small cell lung cancer)
Skin : A375 (malignant melanoma)
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
5/23/2017 |
Inducible expression  low toxicity
Long term expression  drug testing
Easy to scale up
Stable isogenic recombinant (Sir) cell lines
- Downstream applications -
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
Xenografts + in vivo imaging
Screening
Drug
siRNA/miRNA
Compount / miRNA
Echeverri & Perrimon
Nature Reviews Genetics 7, 373-384 (May 2006)
Co-culture / tumor+ stroma cells
Cell-based pathway activity assays
5/23/2017 |
Profiling
Protein (Silac)
RNA / miRNA / CpGs
Protein Quantification / Pathway Identification
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