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04/18/23 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
SMBCa tools
04/18/23 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
ENTRY-Clone
attL1 attL2
gene
KANR
attB1 attB2
gene
AMPR
attR2attR1
AMPR
attL1 attL2
KANR
attR1 attR2
neg. selection
AMPR
Expressionvector
+
SMBCa tools :Gateway™ recombination system
04/18/23 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
attL1 attL2
attR3attR4
ENTRY-Clones
Destination Vector
attL1 attL2
gene
KANR
Promoter
KANR
attL2R attL3
shRNA against UTR
KANR
AMPR
X
X
X
attL4 attL1R
attL4 attL1R
attL2R
attB4Expression Vector
AMPR
attB1 attB2 attB3
attL3
SMBCa tools :Gateway™ multisite recombination
04/18/23 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
ENTRY-Clone
attL1 attL2
gene
KANR
attR1 attR2
neg. selection
AMPR
Expressionvector
~ 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
SMBCa tools :Entry-clone resources
04/18/23 |
Dr. Rainer Will
DKFZ Heidelberg
GPCF / CIA
RTKRTK
PP
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
04/18/23 |
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
CM
V-Pro
SV40 Poly
A
fi ori
Ka
n
R
-P
ro
m
o
te
r
K
H S V P o l y A
2
0
0
0
3
0
0
0
40 0 0
50
00
7000
Acceptor plasmid Acceptor plasmid
Recombination of :
Inducible expression constructs (genes / shRNA/ miRNAs) Resistance gene (Hyg)
attB2 B
G
H
p
A
0 P o l y A
tR
Am
p Resistanc
o
n
st 0
70
00
9530 bp9530 bp
Expression Clone Expression Clone
Stable isogenic recombinant (Sir) cell lines
04/18/23 |
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)
04/18/23 |
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
04/18/23 |
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/miRNA Compount / miRNA
Screening
Profiling
Protein (Silac) RNA / miRNA / CpGs
Co-culture / tumor+ stroma cells Cell-based pathway activity assays