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06/21/22 | Dr. Rainer Will DKFZ Heidelberg GPCF / CIA SMBCa tools

SMBCa tools

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DESCRIPTION

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. - PowerPoint PPT Presentation

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Page 1: SMBCa  tools

04/21/23 |

Dr. Rainer Will

DKFZ Heidelberg

GPCF / CIA

SMBCa tools

Page 2: SMBCa  tools

04/21/23 |

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

Page 3: SMBCa  tools

04/21/23 |

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

Page 4: SMBCa  tools

04/21/23 |

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

Page 5: SMBCa  tools

04/21/23 |

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

Page 6: SMBCa  tools

04/21/23 |

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

Page 7: SMBCa  tools

04/21/23 |

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)

Page 8: SMBCa  tools

04/21/23 |

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

Page 9: SMBCa  tools

04/21/23 |

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