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Victoria Rusakova Senior Scientist Sigma-Aldrich Corporation. Gene Silencing Strategies for Dissecting Disease Pathways. Agenda. Introduction to RNAi shRNA Lentiviral Transduction System Arrayed Kinome shRNA Library - PowerPoint PPT Presentation
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Gene Silencing Strategies for Dissecting Disease Pathways
Victoria Rusakova Senior Scientist
Sigma-Aldrich Corporation
Agenda
• Introduction to RNAi
• shRNA• Lentiviral Transduction System• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell survival
• siRNA• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
2
33
Modulation of Gene Expression
• Small molecules• mAbs• Aptamers
Central Dogma of Molecular Biology
• siRNA• shRNA
RNA Protein
• Zinc finger nucleases
DNA
4
Areas Using RNAi Technology
• Gene function analysis• Testing or verifying predicted gene function
• Pathway analysis• Target the expression of a given gene in a pathway
and monitor the expression of other genes to identify those genes associated with the target gene
• Target identification and validation• Identification of potential drug targets, at the gene
or protein level
• Drug discovery• Develop potential therapeutic compounds based
on identified targets
2006: The Nobel Prize in Physiology and Medicine awarded to Andrew Z.
Fire and Craig C. Mello
RNAi: Types of Interfering RNAs
• Synthetic based • Small or short interfering RNAs (siRNA)
– Transfected directly into cells as oligonucleotides– Do not perpetuate as vectors
• dsRNA molecules (duplexes) shorter than 30bp• Silencing duration and effectiveness mainly regulated
by transfection efficiency
• Clone based • Short hairpin RNAs (shRNA)
– Give rise to siRNA after processing by Dicer protein • Encoded by DNA vectors allowing multiple delivery
methods– Standard transient transfection– Stable transfections– Delivery by virus5
6
RNAi Delivery to the Cell
Agenda
• Introduction to RNAi
• shRNA• Lentiviral Transduction System• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell survival
• siRNA• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
7
8
Recombinant Lentiviral Life Cycle
Viral Transduction Laboratory Workflow
9
Viral Titer and MOI (Multiplicity of Infection)
• Viral titer is a very important factor• Allows determination of the correct experimental conditions using MOI
– MOI (Multiplicity of Infection) used for desired transduction efficiency– The number of transducing lentiviral particles per cell
• When transducing a cell line for the first time, a range of MOI should be tested– Most successful screen require an MOI of 0.5 to 5.0
10
Lentiviral-mediated Gene Transfer in Different Cell Lines
• Significance of controlled conditions in lentiviral vector titration• Use MOI for predicting gene transfer events
Efficiency of lentiviral-mediated gene transfer to commonly used cell lines
under different MOI
Genet. Vaccines Ther. 2(1):6 (2004)
Zhang B., et al., Department of Medicine, University of Queensland, Prince Charles Hospital, Brisbane, Australia
11
Enhancing Transduction Efficiency
• Magnetic transduction• Applying magnetic fields during
transduction to potentiate cell targeting and binding
• Serial transductions• Allow the cells to recover for 1 day
after initial transduction and follow with a second round
• Infecting cells with a higher titer virus• VSV-G envelope protein allows for
concentration via ultracentrifugation and ultrafiltration
VSV-G envelope protein
12
Enhancing Transduction of Primary Cells
TurboGFP particles + polybrene TurboGFP particles + ExpressMag
Human keratinocytes transduced at a MOI of 1, incubated for 45 hours
13
Viral Transduction Laboratory Workflow
14
Transient versus Stable Transduction
Time and cell division affects gene expression
• Gives immediate assessment of the system’s efficiency
MOI 5
HT-29 cells
• Allow to establish clonal stable cell lines
• Provides a system for long-term gene silencing and phenotypic observation
CHO-K1 cells
MOI 1
15
Agenda
• Introduction to RNAi
• shRNA• Lentiviral Transduction System• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell survival
• siRNA• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
16
• Objective: Identify genes that, when silenced, can either enhance or suppress a given phenotype
Modifier Screen
17
• Optimization Plate• Pre-arrayed aliquots of TurboGFP
particles and controls• Ideal for determination of optimal cell
number and MOI for LentiExpress assays
• Human Kinase Plate• A quick method for carrying out kinase
screens• 3109 pre-arrayed lentiviruses
– shRNAs targeting 673 human kinase genes and controls
– A total of 41 96-well plates– Up to 80 shRNAs per plate
LentiExpress Plates
18
Can
cer I
ncid
ence
(per
100
K)
Year
Prostate cancer
Prostate Cancer is the Most Frequently Diagnosed Cancer in American Men
19
normal PIN cancer metastases androgen deathindependence
Progression
Transition to Metastatic Disease
20
LNCaP cells
Gene knockdown + -
Experiment – Knockdown Genes in an Androgen-dependent Cell Line
21
Perc
ent E
xpre
ssio
n
120
100
80
60
40
20
0Untreated H2 H3 H5 H6
Androgen Receptor Knockdown Normalized to Untreated Cells and Cyclophilin
Validation of shRNA Clones in LNCaP Cells
22
LNCaP cells transduced with non-targeting shRNA
LNCaP cells transduced with androgen receptor shRNA
LNCaP Cells Treated with AR shRNA
23
0
20
40
60
80
100
120
4 days 5 days 7 daysTime
% E
xpre
ssio
n
Androgen Receptor
Non-Target
Androgen Receptor Knockdown
Androgen receptor knockdown is stable under experimental conditions of the assay24
Lentiviral shRNA particles targeting kinases
LNCaP Cells
Puromycin selection
Split 1:2shRNA shRNA +
androgen
Viability assay Viability assay
Modifier Screen
25
% Growth Relative to Control -- Vehicle
% G
row
th R
elat
ive
to C
ontr
ol –
And
roge
nshRNA Kinome Screen – LNCaP
26
Agenda
• Introduction to RNAi
• shRNA• Lentiviral transduction system• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell survival
• siRNA• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
27
28
Hypothesis
• Overexpression and activation of specific kinases occurs during growth of osteosarcoma cells
• Disruption of specific kinases will cause osteosarcoma cell death or apoptosis
• These kinases have the potential to be drug targets for sarcoma
28
29
10,000 40,00020,000 80,000 160,000
Various seeding densities (cells/mL) were plated in wells containing tGFP positive control particles
Courtesy of Zhenfeng Duan, M.D.
Determining Optimal TransductionConditions in KHOS
29
30
pLKO.1 Control Particles (C)Non-Target shRNA Control Particles (N) Control Media (M)
1 µg/ml of puromycin causes complete cell death of KHOS, U-2OS and UCH1 in 5 days
Courtesy of Zhenfeng Duan, M.D.
Negative Controls Used in the Optimization Plate
30
Protocol for shRNA Kinase Screen in Human Osteosarcoma Cells
31
Replace wells with fresh media
Incubate plate at 37 °C, 5% CO2
overnight
Add puromycin- supplemented
media at 1µg/mL
Dispense KHOS cells into 96 well lentiviral shRNA
kinase platesRemove plates from incubator
Analyze results with a cell proliferation
assay kit
Courtesy of Zhenfeng Duan, M.D.
overnight
Incubate plate at 37 °C, 5% CO2
7 daysChange media every 2
days with puromycin
Incubate plate at 37 °C, 5% CO2
32
C*C C
C
N
N
N
N
M
M
M
M
M
M
M
M
A7 A8 A9 A10 A11
B11
C2 C3 C4 C5
M
Courtesy of Zhenfeng Duan, M.D.
Positive Hits from Screen
32
Positive Hit 1: PLK1Reduced Viability Upon Silencing
pLKO.1 particles
Non target particles
Media control
00.10.20.30.40.50.60.70.80.9
G7 G8 G9 G10 A12(C)
C12(N)
H12(M)
Abs
orba
nce
(490
nM
)
33
Positive Hit 2: ROCK1Reduced Viability Upon Silencing
0.00
0.25
0.50
0.75
1.00
B11 C2 C3 C4 C5 A12(C)
C12(N)
H12(M)
Abs
orba
nce
(490
nM
)
pLKO.1 particles
Non target particles
Media control34
35
LentiExpress Kinase Screen Summary
• Identified 4 gene candidates as potential therapeutic targets in osteosarcoma cells, including PLK1 and ROCK1
• KHOS osteosarcoma cells exhibited decreased cell proliferation upon knockdown of these genes
35
Agenda
• Introduction to RNAi
• shRNA• Lentiviral transduction system• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell survival
• siRNA• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
36
RNAi: Types of Interfering RNAs
• Synthetic based • Small or short interfering RNAs (siRNA)
– Transfected directly into cells as oligonucleotides– Do not perpetuate as vectors
• dsRNA molecules (duplexes) shorter than 30bp• Silencing duration and effectiveness mainly regulated by
transfection efficiency
• Clone based • Short hairpin RNAs (shRNA)
– Give rise to siRNA after processing by Dicer protein • Encoded by DNA vectors allowing multiple delivery
methods– Standard transient transfection– Stable transfections– Delivery by virus37
MISSION esiRNA Technology
Transfect into cell
“Super-pool” of hundreds of siRNAs against 1 target gene
Assembly into RISC
Targeting of single mRNA
mRNA cleavage and degradation38
Generation of esiRNA
39
MISSION esiRNA
esiRNA Gene #1
esiRNA Gene #2
esiRNA Gene #3
esiRNA Gene #4
etc.
1 esiRNA super-pool targeting one gene per well
40
41
Discovering Modulators of Embryonic Stem Cell Identity
• Objective• Obtain a systematic understanding of the genes associated with ESC identity
• Approach• Perform a genome-scale RNAi screen to identify genes regulating ESC identity
using an Oct4 reporter assay
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
42
Oct4 Assay
• Oct4 expression can be used to monitor the differentiation status of ESC
• Screen performed in an Oct4 reporter mouse embryonic stem cell line (Oct4-Gip) • GFP expression is controlled by Oct4 regulatory elements
• Transfect cells with esiRNA and monitor changes in GFP expression
• Quantification of GFP fluorescence faithfully reflects the self-renewal and differentiation status in individual cells
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
43
Oct4 Assay: Proof of Principle
GFP Expression• Individual wells transfected with• Control luciferase esiRNA• esiRNA to known pluripotency
regulators– Sox2– Oct4– Stat3
• Visualized GFP by microscopy or FACS analysis
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
44
Overview of Oct4 High-throughput Assay
Transfect Oct4-Gip ESC with control or genome-
scale esiRNA library
High-throughput GFP fluorescence readout to
identify primary hits
Negative control (Luciferase esiRNA)
Primary hit (cells have reduced GFP)
No hit or negative control (cells have
high GFP Expression)
Primary hit or positive control
(cells have reduced GFP Expression)
Readout
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
45
Summary of Oct4 High-throughput Assay
• 259 known and novel candidate pluripotency genes identified
• Secondary screen performed using individual esiRNAs synthesized for the 21 strongest candidates• 16 genes were confirmed
• Validated targets included components of the of the Pol II-associating factor 1 complex (Paf1C)• Paf1C contains Paf1, Ctr9, Cdc73, Rtf1, and Leo1• Regulates transcription initiation, elongation, and start site selection
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Paf1C Affects the Expression of Pluripotency and Lineage-marker Genes
46Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Summary of Study
• siRNA (esiRNA) is an effective tool for modulating gene function in stem cells
• A screen using esiRNA identified 259 known and novel candidate pluripotency genes
• Validated targets included components of the of the Pol II-associating factor 1 complex (Paf1C)
• Paf1C affects the expression of pluripotency and lineage-marker genes
47
48
49
Review of RNAi Effectors
siRNA shRNA
• Benefits• Simple • Titratable• Modifications available• Pooling is straightforward• Efficiently transfected
– Easy to transfect cell lines
• Disadvantages• Hard to transfect cells• Transient knockdown• Non-renewable
• Benefits• Renewable resource• Transient or stable knockdown• Transfection or viral delivery
– Viral delivery to most cells• In vivo use potential
– Knockdown mice
• Disadvantages• Design rules less understood• Transfection less efficient
50
• Goals• Create a lentiviral based shRNA libraries targeting human and mouse genes• Make clones available to researchers worldwide for the study of disease and gene
function
• Academic Laboratories• Broad Institute, MIT/Harvard, Massachusetts General Hospital, Dana Farber Cancer
Institute, Whitehead Institute, Washington University and Columbia University
• Life Science Organizations• Sigma-Aldrich, Novartis, Eli Lilly, Bristol-Myers Squibb and Academia Sinica in Taiwan
The RNAi Consortium (TRC)
51
TRC1 shRNA Transfer Vector
• Transfer vector• pLKO.1-puro• Lentiviral-based (HIV derived) Vector
• shRNA Promoter• U6 (human)
• Design• Broad Institute algorithm• 21 bp stem• 6 bp loop
• 5 clones per target gene• High gene coverage• Multiple knockdown levels• Verification of phenotype
–Different shRNA produces same result
• 3' UTR clone for cDNA rescue
52
TRC2
• TRC2 Goals• KD evaluation for 150,000 clones
by qRT-PCR• Optimize vector elements • Consider and evaluate special
purpose vectors• Develop new and improved
screening methods– Pooled libraries
53
TRC2 shRNA Transfer Vector
Woodchuck hepatitis post-transcriptional regulatory element (WPRE)
• Sigma uses 3rd generation safety & design
• SIN vector (self inactivating vector)
• Replication incompetent lentiviral particles
• Recommended biosafety level: BSL-2