latest state of the art rnai screening · holger erfle viroquant-cellnetworks rnai screening...
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Holger Erfle
ViroQuant-CellNetworks
RNAi Screening Facility
BIOQUANT
University Heidelberg
26.05.2011
Latest state of the art RNAi screening
• RNAi Screening in BioQuant
• Examples of RNAi screens
• Outlook
Outline:
• RNAi Screening in BioQuant
Technology
Platform
Interaction
of viral and
cellular Systems
Modeling
Platform
ViroQuantSystems Biology of Virus-Host Cell interactions
Genome-wide screen for host cell factors
essential for viral replication
Reconstructing signaling networks
e.g. data storage, management andanalysis
Assay development
siRNA library
Automated transfection
Image acquisition
Image analysis
3-12 months
Major components of an RNAi Screen
Assay development
siRNA library
Automated transfection
Image acquisition
Image analysis
Major components of an RNAi Screen
Gene specific degradation of target mRNA in the cells
Loss of function phenotype
Transfection into mammalian cells: Hela, MCF7, Prim. Melanoma, Prim Neurobl.
20.273 human genes ɬ 3 siRNAs, 60819 siRNAs (Ambion, ABI)
Incubation time
RISC
RNAi of protein coding genes
Assay development
siRNA library
Automated transfection
Image acquisition
Image analysis
Major components of an RNAi Screen
Pipetting robotHamilton “Star”
DNA PrinterBio- Rad „Chipwriter Pro“
Automated transfection
siRNA\miRNA\cDNA
complexed
with transfection solution
Cell Arrays
Cells
Ziauddin J. and Sabatini D.M., Nature. 2001
ready to transfect 96/384 well plates(H.Erfle et al., JBS, 2008)
ready to transfect Cell Arrays (384 spots)Erfle et al., Nat. Prot. (2) 2007
Pipetting robotHamilton “Star”
DNA PrinterBio- Rad „Chipwriter Pro“
AssaysHIV HCV DV PV AAV UPS PC1 LDL etc. Kräusslich Bartenschlager Marchini Kleinschmidt Nickel Starkuviene Runz
Runz: (Bartz, Cell Metab., 2009) Nickel: (Ebert, Traffic, 2010) Bartenschlager: (Reiss, Cell Host Microbe, 2011)
Automated transfection
Assay development
siRNA library
Automated transfection
Image acquisition
Image analysis
Major components of an RNAi Screen
2 Leica SP5 confocal systems3 Olympus Biosystems ScanR wide-field systems
•Motorized stage
•Automated focus
•Automated image storage
Automated screening microscopes
Assay development
siRNA libary
Automated transfection
Image acquisition
Image analysis
Commercial software, e.g. ScanR analysis,
Open source: ImageJ ,Cell Profiler
Collaboration: Rohr, Hamprecht, Hesser
Major components of an RNAi Screen
LSDFM. Hemberger
LSDFM. Hemberger
RNAi Screening Facility
RNAi Screening Facility
Automated transfection
10 – 20.000 genes
30 – 60.000 siRNAs
RNAi Screening Facility
RNAi Screening Facility
Automated sceening microscopes
K. RohrF. HamprechtJ. Hesser
K. RohrF. HamprechtJ. Hesser
Image processing
Statistics
L. KaderaliL. Kaderali
Data Base
R. SchneiderL. KaderaliMetaCore
R. SchneiderL. KaderaliMetaCore
Bioinformatics
J. EilsC. LawerenzR. Kappe
J. EilsC. LawerenzR. Kappe
1 GBit
• Examples of RNAi screens
Boerner K. et al Biotechnol. J, 2010
RNAi screening platform to identify host factors involved in
HIV-1 (HCV, DV, AAV, PV) replication
data storage druggable genome-wide screens (27309 siRNAs): 3 TB
collab.: K. Rippe
Osterwald S. et al, Biotechnology J. 2011
promyelocytic leukemia (PML) nuclear bodies
3 TByte
educated guess screen ~ 100 siRNAs
Elucidate the alternative
lengthening of telomeres pathway
Lisauskas T, and Starkuviene V. Methods Mol Biol. 2011
Cell arrays for the measurement of organelle dynamics in living cells
parallel transfection with cDNA
parallel stimulus - BFA destabilizes Golgi complex
data aquisition: each sample 1 images/ 1 min over 30 min
(potential) data storage genome-wide screen: 300 TB
Time resolution:30 min.
Imaging time:72 hours starting
24 after seeding
Expected data volume:Genome-wide: 450 TByte
Westermann F., König R.
Neuroblastoma Screen for genes whose knock-down lead to mitotic and cell death effects
•Outlook
2) Regulatory networks of miRNAs
An integrated high throughput and super high
resolution microscopy platform for analysing
miRNA targets in living cells
Funding: Baden-Württemberg Stiftung
New Methods for LifeSciences
Funding period: 2010-2015
1) RNAi of ncRNAs
Functional analysis of non-coding RNAs
in living cells
Funding: BMBF (SysTec)
New Methods for Systems Biology
Funding period: 2009-2012
Novel tools for systems biology
Reymann J. et al., BioTechniques, 2009
in Kollaboration mit:
• Graffinity PharmaceuticalsGmbH
500µm Glassubstrat
96 × 96 = 9216 Spots20µm
200nm Titan-Beschichtung
100µm
siRNA – PLK Hoechst
Whole genome „Cell arrays“
resolution
thro
ughput
High-throughput
Confocal Screening
Super-resolution (STED,STORM)
2D resolution
3- 600 TByte
3D structure-resolution
3- 600 TByte
z
x
y
y
x
3D structure-resolution
3 – 600 TByte
y
200µm
20µm
y
x
z
Confocal STED, STORM
image processing 1 image processing 2 image processing 3
X.Y position
X.Y position
Correlative Microscopy
ImageJ
...
STRING?
DAVID?
...
OP - Data
Mining
Open source software based data mining - based on KNIME
L. Kaderali
...
KNIME
data knowledge
Acknowledgements
Funding:
RNAi Screening Facility
Screening core Nina Beil
Jürgen Beneke
Development Jürgen Reymann
Manuel Gunkel
Slawek Mazur –HMLS-
Johannes Rittinger
all collaboration partners