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RNA Interference. Iain Fraser. Initiation step. (RNA-induced silencing complex). Dicer. Effector step. Model for RNAi mechanism. Hammond et al., 2001. U6 Expression Cassette H1 Expression Cassette . +1. +1. U6 pr. G-N18- TT-loop -N’18-C. TTTT. H1 pr. N19- TT-loop -N’19.

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rna interference

RNA Interference

Iain Fraser

model for rnai mechanism

Initiation step

(RNA-induced silencing complex)

Dicer

Effector step

Model for RNAi mechanism

Hammond et al., 2001

expression of sirnas from pol iii promoters

U6 Expression Cassette H1 Expression Cassette

+1

+1

U6 pr.

G-N18-TT-loop-N’18-C

TTTT

H1 pr.

N19-TT-loop-N’19

TTTT

Pol III

Pol III

N19

N19

UU

UU

5’-

5’-

Short hairpin

RNA

N’19

N’19

UU

UU

Processing

Processing

N19

N19

UU

UU

5’-

5’-

siRNA

duplex

-5’

-5’

N’19

N’19

UU

UU

Expression of siRNAs from pol III promoters
target sequence selection
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

4 linkers designed

per gene

ccdB counter-selection

prevents background

in cloning step

Hairpin

Linker

+

pEN_mH1c

BamH1

Xho1

attL1

mH1

ccdB

attL2

YFP

Gene

attL1

mH1

attL2

Co-express hairpins with YFP tagged gene of interest to identify effective hairpin

Vector design
is data from transient expression reliable

188

98

Zeo control

PTEN -C

PTEN -D

PTEN -A

PTEN -B

62

49

38

28

17

14

Is data from transient expression reliable?
  • Best hairpin against heterologously expressed gene is also most effective against endogenous gene after selection of transfected cells

Hairpins

Control

A

B

C

D

YFP-PTEN

PTEN

Transient transfection

Zeocin selection

efficacy of cxcr5 syk and galpha i2 hairpins

CXCR5-YFP

YFP-Syk

YFP-Gi2

M

A

B

C

D

M

X

A

B

C

D

Hairpins:

M

A

B

C

D

IB: anti-YFP Ab

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
subcloning of sirna cassettes into lentiviral constructs

Hairpin

pEN_mH1c

attL1

mH1

attL2

+

pDSL_UGIP

LTR

FLAP

attR1

attR2

Ubi-C

GFP

IRES

Puro

WRE

LTR

LR site specific

recombination reaction

pL_hp-UGIP

LTR

FLAP

attB1

attB2

Ubi-C

GFP

IRES

Puro

WRE

LTR

mH1

Hairpin

Subcloning of siRNA cassettes into lentiviral constructs
genomic pcr confirms integration of pol iii cassettes

CXCR5 siRNA stable

Syk siRNA stable

CXCR5 vector

Syk vector

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
integrated sirnas have no effect on target mrna in transduced wehi cells
Integrated siRNAs have no effect on target mRNA in transduced WEHI cells

Quantitative

PCR

Microarray

targeting of g alpha i2 in j774a 1 monocytes

Gi2 primer

Gi2 siRNA

Gi3 siRNA

Gi1 siRNA

Control

1.2

1.0

0.8

Blot: anti-Gi2

Expression of mRNA(normalized with GAPDH)

0.6

0.4

Blot: anti-tubulin

0.2

0.0

Gi3

Control

Gi2

SiRNA

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

mRNA

Protein

Jong-Ik Hwang, Simon Lab

reporter assay for assessment of rnai capacity

GFP-Luc

lacZ

si-lacZ

CMV

CMV

pol III

Target cell

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
rnai reporter assay wehi231
RNAi reporter assay: WEHI231

Beta-Gal Activity, RU

+-

++

++-

++

LacZ

siLacZ

++++

rnai reporter assay j774a 1 and raw264 7

RAW 264.7

B-Gal Activity, RU

+-

+ +

+ ++

+ +++

+ ++++

LacZ

siLacZ

J774A.1

B-Gal Activity, 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
rnai reporter assay 3t3l1 ic 21 and n1e 115

3T3L1

IC-21

B-Gal Activity, RU

B-Gal Activity, RU

+-

++

+ ++

LacZ

siLacZ

+-

++

+ ++

LacZ

siLacZ

N1E-115

B-Gal Activity, RU

+-

++

+ ++

LacZ

siLacZ

RNAi reporter assay: 3T3L1, IC-21 and N1E-115
lentiviral mediated rnai in raw264 7 cells 1
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
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
Acknowledgements

Molecular Biology Lab

Joelle Zavzavadjian

Pamela Eversole-Cire

Jamie Liu

Dan Allen

Mei Wang

Sangdun Choi

Assay Lab

Tamara Roach

Caltech

Jong-Ik Hwang

Melvin Simon

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