rna interference iain fraser. model for rnai mechanism hammond et al., 2001 dicer (rna-induced...
TRANSCRIPT
Model for RNAi mechanism
Hammond et al., 2001
Dicer
(RNA-induced silencing complex)
Initiation step
Effector step
Expression of siRNAs from pol III promoters
H1 pr. N19-TT-loop-N’19 TTTT
+1
UU
UU
Pol III
5’-
UU
N’19UU
5’-
-5’
Processing
Short hairpin RNA
siRNA duplex
N’19
U6 pr. G-N18-TT-loop-N’18-C TTTT
+1
UU
UU
Pol III
5’-
UU
N’19UU
5’-
-5’
Processing
N’19
U6 Expression Cassette H1 Expression Cassette
N19N19
N19N19
Target sequence selection
• Length: 19 basepairs• Original Tuschl rule of selecting N19 immediately
following a AA is not necessarily observed in our design
• %GC content: 45-55%, optimum 50%• Tm: 45-65oC, optimum 55oC• Avoid AA at start and TT at end of sequence to
prevent premature termination• BLAST to ensure specificity• We find selection of an effective target sequence
to be arbitrary
Vector design
attL1 mH1 ccdB attL2
BamH1 Xho1
HairpinLinker
pEN_mH1c
attL1 mH1 attL2
+
4 linkers designed per gene
ccdB counter-selection prevents background in cloning step
YFP Gene
Co-express hairpins with YFP tagged gene of interest to identify effective hairpin
Is data from transient expression reliable?
• Best hairpin against heterologously expressed gene is also most effective against endogenous gene after selection of transfected cells
YFP-PTEN
PTEN
A B C DCon
trol Hairpins
98
62
49
38
281714
188
PTE
N -A
PTE
N -B
PTE
N -D
Zeo
cont
rol
PTE
N -C
Transient transfection Zeocin selection
RNAi target genes
Gene Status Gene Status Gene Status
PTEN Complete G alpha i1 Complete PI3K p110g Complete
Jnk1 Complete G alpha i2 Complete PDK1 Complete
G alpha 13 Complete G alpha i3 Complete CXCR5 Complete
G alpha 12 Complete G alpha q Complete Syk Complete
G/C/YFP Complete G beta 1 Complete PI3K p110d In progress
G beta 2 Complete PI3K p85a In progress
G beta 3 Complete CD19 Complete
G beta 4 Complete SHIP In progress
G beta 5 Complete CXCR4 In progress
RNAi target genes
Gene Status Gene Status Gene Status
PTEN Complete G alpha i1 Complete PI3K p110g Complete
Jnk1 Complete G alpha i2 Complete PDK1 Complete
G alpha 13 Complete G alpha i3 Complete CXCR5 Complete
G alpha 12 Complete G alpha q Complete Syk Complete
G/C/YFP Complete G beta 1 Complete PI3K p110d In progress
G beta 2 Complete PI3K p85a In progress
G beta 3 Complete CD19 Complete
G beta 4 Complete SHIP In progress
G beta 5 Complete CXCR4 In progress
Efficacy of CXCR5, syk and Galpha i2 hairpins
• Hairpins and YFP tagged gene were used to co-transfect P19 cells• Cells were harvested 48hr post-transfection• Mock control was vector containing hairpin against different gene• X control in Galpha i2 experiment was chemically synthesized siRNA
duplex directed against Galpha i2
M BA C D
YFP-Syk
M BA C D
CXCR5-YFP
Hairpins: M BA C D
YFP-Gi2
X
IB: anti-YFP Ab
Subcloning of siRNA cassettes into lentiviral constructs
pEN_mH1cattL1 mH1 attL2
Hairpin
attR1 attR2FLAPLTR Ubi-C WREGFP LTRIRES Puro
pDSL_UGIP
attB1 attB2FLAPLTR Ubi-C WREGFP LTRIRES PuromH1
Hairpin
pL_hp-UGIP
+
LR site specific recombination reaction
Genomic PCR confirms integration of pol III cassettes
• PCR carried out with sense primer in H1 promoter upstream of hairpin and antisense primer in ubiquitin promoter downstream of hairpin
• PCR products excised from gel and sequenced
• Sequence confirms both Syk and CXCR5 hairpins integrated in stable lines
Syk
siR
NA
stab
le
Syk
vect
or
CXC
R5
siR
NA
stab
le
CXC
R5
vect
or
Integrated siRNAs have no effect on target protein in transduced WEHI cells
98
62
Wt W
EHI c
ontro
lS
yk-s
iRN
A
Syk western98
62
Wt W
EHI c
ontro
lS
yk-s
iRN
A
Syk western
CXCR5 FACS Data
Syk Western Blot
Integrated siRNAs have no effect on target mRNA in transduced WEHI cells
ReadoutNormalized mRNA Expression
Level
Syk expression in WEHI control 0.798
Syk expression in Syk-siRNA stable WEHI 1.002
CXCR5 expression in WEHI control 0.541
CXCR5 expression in CXCR5-siRNA stable WEHI 0.534siRNA Target
Syk expression in WEHI control
Syk expression in siRNA stable WEHI
Signal intensity Background Signal intensity Background
Syk 20823 1002 32539 1361
CXCR5 20851 1492 20791 1944
siRNA Target
CXCR5 expression in WEHI control
CXCR5 expression in siRNA stable WEHI
Signal intensity Background Signal intensity Background
CXCR5 1518 1305 2462 1781
Syk 1529 1455 2420 2167
Quantitative PCR
Microarray
Targeting of G alpha i2 in J774A.1 Monocytes
• Cells infected with lentivirus containing Galpha i2-C hairpin• Same UGIP lentiviral backbone as used for Syk and CXCR5
viruses• Data from puromicin selected cells
Control Gi2SiRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Exp
ress
ion
of m
RN
A(n
orm
aliz
ed w
ith G
AP
DH
) Gi2 primer
Gi3 Blot: anti-tubulin
Blot: anti-Gi2
Gi2
siR
NA
Gi3
siR
NA
Gi1
siR
NA
Con
trol
mRNA Protein
Jong-Ik Hwang, Simon Lab
Reporter assay for assessment of RNAi capacity
• Assay for lacZ and luciferase activity 48hr post-transfection• Assay is very sensitive, giving data from <5% transfection
efficiency
Target cell
CMV GFP-Luc
CMV lacZ
pol III si-lacZ
RNAi reporter assay: J774A.1 and RAW264.7
• Transfection efficiency of J774s very low• Required electroporation to achieve detectable
lacZ activity• LacZ activity at lower limit of assay sensitivity
+-
RAW 264.7
+ +
+ ++
+ +++
+ ++++
B-G
al A
cti
vit
y,
RU
LacZ
siLacZ
J774A.1
+-
++
+ ++
B-G
al A
cti
vit
y,
RU
LacZ
siLacZ
RNAi reporter assay: 3T3L1, IC-21 and N1E-115
IC-21
+-
++
+ ++
B-G
al A
cti
vit
y,
RU
LacZ
siLacZ
N1E-115
B-G
al A
cti
vit
y,
RU
LacZ
siLacZ
+ ++
+-
++
B-G
al A
cti
vit
y,
RU
3T3L1
+-
++
+ ++
LacZ
siLacZ
Lentiviral-mediated RNAi in RAW264.7 cells:1
• Three siRNA hairpins designed against TREM-2B receptor
• siRNA-expressing lentivirus used to transduce RAW264.7 cell line containing stably expressed FLAG-TREM-2B
• siRNA efficacy assessed by FACS detection of FLAG tag
Tamara Roach, AfCS Assay Lab
Summary/Conclusions
• We have established a flexible vector-based system for the development of effective siRNAs against genes of interest to the AfCS.
• Expression of such siRNAs from lentiviral vectors permits the transduction of hematopoietic cell lines.
• WEHI231 cells transduced with siRNAs do not exhibit RNAi-mediated target knockdown. It remains unclear whether this is due to the siRNAs not being expressed from pol III promoters, or the absence of the components of the RNAi machinery required to recognize the siRNAs.
• Vector-based RNAi was effective in six other cell lines tested by reporter assay: J774A.1, RAW264.7, IC-21, N1E-115, 3T3-L1 and HEK293.
• Lentiviral-mediated RNAi was effective in both J774A.1 and RAW264.7 cells