RNA Interference

Iain Fraser

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

Infection of WEHI231 cells with siRNA-CXCR5 and siRNA-syk lentiviruses

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: WEHI231B

eta

-Gal A

cti

vit

y,

RU

LacZ

siLacZ

+-

++

++-

+++++

+

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

Lentiviral-mediated RNAi in RAW264.7 cells: 2

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

Acknowledgements

Molecular Biology Lab

Joelle ZavzavadjianPamela Eversole-CireJamie LiuDan AllenMei Wang

Sangdun Choi

Assay Lab

Tamara Roach

Caltech

Jong-Ik HwangMelvin Simon