Long term RNA interference :

The first library of stable human knock down cells

Denis BiardCEA. DSV-iRCM, Fontenay-aux-Roses 92265, France

State of the art

Strategy of “single knock down”

Strategy of “double knock down”

Examples of targeted human genes (over 130)

Conclusions

Example of « Loss of function »

The 1st library of « silenced » clones for DNA repair genes

2009

Pol iota (NM_007195

Pol eta (NM_006502)

Pol Mu (NM_013284)

Pol Kappa (NM_016218)

Pol theta (polQ (NM_199420)

Pol Lambda (NM_013274)

BACH1 (NM_032043)

Pol Beta (NM_002690)

Rev1 (NM_016316)

TNF-R2 (NM_001066)

TRAIL-R2 (NM_147187)

FADD (NM_003824)

FAS (NM_152877)

Apoptotic receptors

Since 2002, RNA Interference (RNAi) has become the best way for studying gene function by

reverse genetic approaches. Most studies describing RNAi in human cells are based on transient

transfection of siRNA duplexes or plasmids, or transient infection of virus-expressing siRNA

(small interfering RNA) constructs. However, integrative plasmids or viruses can lose the

expression of the siRNA cassette during selection pressure and long term culture.

To overcome these shortcomings, I have designed replicative plasmids driving the in cellulo

siRNA synthesis through shRNA (small hairpin RNA) sequences. I have harnessed the efficiency

of the shRNA approach with Epstein–Barr virus (EBV)-derived vectors (CEA patent, 2005) in

order to establish a unique cell model in which one specific gene was silenced for an

undetermined period of time. Since these vectors behave like endogenous transcription units, it

was expected that they would tightly regulate siRNA expression over a long period of time. At

present, with more than 700 pEBVsiRNA vectors targeting 130 human genes in diverse

metabolic pathways (e.g. DNA repair, oxydative stress, or cell cycle control), I have

demonstrated that pEBVsiRNA vector greatly enhanced long-term gene silencing in cultured

human cells. About 40 stable clones have been analyzed for their loss of function.

Importantly, our shRNA sequences are now designed with the DSIR program (Y.

Vandenbrouck; CEA) including an exact similarity search algorithm for potential off-target

detection.

The combination of RNAi technology, DSIR program and pEBV-derived vectors offers an

exceptional opportunity to rapidly create a set of stable knock-down clones in numerous cell

lines, covering various fields of genetics. We could mimic human diseases in a comparable

genetic background. This approach may have several applications in drug screening and in the

early stages of testing new therapies.

This library of silenced clones, which evolves constantly, is a powerful tool for Basic

Research, for Diagnostic and for the Screening of new drugs.

These cell lines, now termed SilenciX cells, are available to the scientist community through

tebu-bio (http://www.tebu-bio.com/file/thematic/92/).

pEBVsiRNA-based long term gene silencing efficiencies are successfully assessed in different

human tumor-derived cell lines, such as HeLa, RKO, HCT-116, MRC5-V2, U2OS, MCF7,

Caco2 or SH-SY5Y cells. In well-polarized and differentiated intestinal Caco2 cells, such an

approach could improve drug screening.

In our strategy of “single knock down” (one gene is silenced), three vectors per gene coding different siRNA

sequences, are constructed and validated. The rate of gene silencing is usually > 80%. Thereafter, clones are

isolated randomly and analyzed for several months in order to assess their loss of function.

For XRCC1, the three vectors gave rise to very efficient gene silencing 4 days after transfection. The pBD1065

vector was selected and different HeLa clones were established. 125 days later, the clone number 3 displayed an

undetectable XRCC1 protein level correlated with a fall down of its partner (ligase III). A similar result was

observed when the Ligase III gene was targeted first.

1. LigIIIKD cl.6 (day40)

2. LigIIIKD cl.11 (day40)

3. LigIIIKD population (day40)

4. CSBKD cl.21 (day120)

5. CSBKD cl.22 (day120)

1 2 3 4 5

wb548

CSB

LigIII

NBS1

PCNA

LigIV

1. DNA-PKcsKD cl. 1 (day 227)

2. Ku70KD (day 5)

3. XRCC4KD cl.9 (day 206)

4. LigIVKD cl. 1 (day 40)

5. LigIVKD cl. 3 (day 40)

6. LigIVKD cl. 6 (day 40)

7. LigIVKD cl. 7 (day 40)

8. LigIVKD cl. 10 (day 40)

9. LigIVKD cl. 12 (day 40

wb549

1 2 3 4 5 6 7 8 9

PCNA

XRCC4

Ku70

NBS1

LigIV

Herein, LigIVKD and LigIIIKD clones, already characterized, are transfected with the pEBVsiPARP1-puro vector

in order to create « double KD » LigIVKD/PARP1KD and LigIIIKD/PARP1KD (hygromycine B + puromycine).

Example of “silent”clones

Amé et al, J Cell Sci (2009)

To create this first library of knock down clones, I have targeted several genes of the main DNA repair

processes and of crucial DNA damage signaling.

Efficient at long term Efficient but lethal In evaluation

MSH2 (NM_000251)

MLH1 (NM_000249)

XPA (NM_000380)

XPE (DDB1) (NM_001923)

XPC (NM_004628)

hHR23A (NM_005053)

hHR23B (NM_002874)

CSA (NM_000082)

CSB (NM_000124)

XPF (NM_005236)

ERCC1 (NM_001983)

XPG (NM_000123)

Ligase I (NM_000234)

Ligase III (NM_013975)

XRCC1(NM_006297

PIF-1 (NM_025049)

FEN-1 (NM_004111)

Ogg1 (NM_016819)

WRN (NM_000553)

BLM (NM_000057)

Rad51 (NM_002875)

Rad54 (NM_003579)

Rad52 (NM_002879)

PARG (NM_003631

PARP1 (NM_001618)

PARP2 (NM_005484)

PARP3 (NM_001003931)

PARP9 (NM_031458)

PARP14 (NM_017554)

Ku70 (NM_001469)

Ku80 (NM_021141)

DNA PKcs (NM_006904)

XLF-Cernunnos (NM_024782)

Ligase IV (NM_002312)

XRCC4 (NM_022550)

Artemis (NM_001033855)

p53 (NM_000546)

p21 (NM_000321)

Rb (NM_000321)

BRCA1 (NM_007295)

BRCA2 (NM_000059)

MRE11 (NM_005590)

Rad50 (NM_005732)

NBS1 (NM_002485)

ATM (NM_000051)

ATR (NM_001184)

FancD2 (NM_033084)

SAF-A (NM_031844)

ASF1A (NM_014034)

ASF1B (NM_018154)

HIRA (NM_003325)

CABIN1 (NM_012295)

Tip60-1 (NM_006388)

TRRAP (NM_003496)

Drosha (NM_013235)

Pol Delta (NM_002691)

SAF-A (NM_031844)

kin17 (NM_012311)

RORa (NM_002943)

HNF4 γ (NM_004133)

HNF4 α (NM_000457)

STOX (NM_152709)

MDR1 (NM_000927)

PATCHED (NM_000264)

hTERT (NM_003219)

TRF1 (NM_017489)

TRF2 (NM_005652)

hTERC (NM_001566

Dyskerin (NM_001363)

Rad51D (NM_002878)

Tankyrase 1 (NM_003747)

Tankyrase 2 (NM_025235)

HPV18 E7 (X04773)

HPV18 E6 (A06328)

VE-Cadherin (NM_001795)

Moesin (NM_002444)

Myosin X (NM_012334)

Tubulin γ (NM_001070)

Rad18 (NM_020165)

CHK2 (NM_007194)

CHK1 (NM_001274)

Wip1 (NM_003620)

Cdc25B (NM_021873)

CDK5 (NM_004935)

p35 (NM_003885)

MAPK2 (NM_002690)

NADPH oxydase 1 (NM_007052)

Thioredoxin 1 (NM_003329)

Thioredoxin 2 (NM_012473)

Glutathione reductase (NM_000637)

Peroxiredoxin 1 (NM_002574)

Peroxiredoxin 2 (NM_005809)

Thioredoxin reductase (NM_182743)

SOD2 (NM_000636)

BER-NER

Signaling

Chromatin

Oxydative MetabolismCell cycle control

Cytosquelet

Adhesion

Maintenance

Télomères

PARP family

HR

TLS

MMR

RecQ helicase

Others

Viral proteins

NHEJ

We aim to knock down two genes in a same cell line. We propose to silence one gene, to characterize clones

and afterwards target a second gene for creating “double knock down” cells. In this way, we can analyze

sequentially the loss of function related to the first and the second gene silencing.

Whenever possible, the loss of a specific protein is correlated with a loss of function. Example for PARP1KD and

PARGKD clones :

07bd0559 07bd0559

07bd0561 07bd0561

07bd0566 07bd0566

Control population

BD650 (x315)

ATMKD

BD1177/11; day 65

(x315)

ATMKD

BD1177/10; day 65

(x315)

DAPIATM

The next step will be to develop this

approach in primary human and rodent

cells in order to accelerate drug discovery

and therapeutic development.

For this purpose, new vectors are in

evaluation in order to detect and follow

transfected cells (e.g. pEBVsiRNA-

CAG2eGFP).

Literature:

Most of these genes are involved in human syndromes or cancers.

Signaling

FancD2Recombination (late S / G2)

Homologous recombination repair (HR)

Single strand annealing (SSA)

Post-replicative Repair

Pre-replicative Repair

Base Excision Repair (BER) Nucleotide excision repair (NER)

Recombination (mainly G0-G1)

Non homologous DNA end joining (NHEJ)

Microhomology mediated end joining (M-NHEJ)

NER (Global Genome Repair)

NER (Transcription coupled repair)

Translesion synthesis (TLS)S Phase

Rad54Rad52

PARP1, PARP2, PARG

XPA XPChHR23AhHR23B

DNA PKcs XRCC4

LigIV

Ogg1

cdc25b MRE11, Rad50, NBS1

BRCA1, BRCA2

BRCA1, BRCA2, ATM, ATR, Chk2, Kin17, FancD2, p53, BLM

ERCC1

XRCC1LigIII

LigI

XPF

Mismatch repair (MMR) MSH2

XLF

BACH1/FancJ, FancD2

pol. Eta, Iota, Mu, Lambda, Béta

Ligase3 DAPI

LigIIIKD clone (partner of XRCC1) 145 days in culture

Population (day 4; 3 vectors)

XRCC1KD clone (partner of LigIII)125 days in culture

XRCC1 DAPI

PARP1KD

Loss of function

Godon et al NAR (2008)

PARP1KDControl PARP2KD

Immunodetection of PAR (10H)

Tritiated NAD incorporation

Lepage F. (CEA)

SOD2 DAPI

BD1673 population (day 35)

BD650 (Control)

09BD134 09BD134

09BD140 09BD140

U2OS cells

3’UTRORF

BACH1 = BRCA1-INTERACTING

PROTEIN 1 (BRIP1) = FANCJ GENE

Betous et al Mol Carcinog (2008)

Pennarun et al. (2008) Nucleic Acids Res. 36, 1741-1754.

Wu et al. (2007) J. Biol. Chem. 282, 31937-31943

Biard and Angulo (2007) Nova Science Publishers, Inc.

Despras et al. (2007) Cancer Res. 67: 2526-2534.

Biard (2007) Nucleic Acids Res. 35:3535-50.

Biard et al. (2005) Mol. Cancer Res. 3, 519-529.

Rey et al. (2009) Mol Cell Biol in press

Amé et al. (2009) J Cell Sci in press

Amine et al. (2009) PLoS ONE, 4:e5018

Bétous et al. (2008) Mol Carcinog. 2008 48, 369-378

Schulte et al. (2008) DNA Repair, 7, 1757-1764

Godon et al. (2008) Nucleic Acids Res., 36, 4454–4464.

Aressy et al. (2008) Cell Cycle, 7, 2234-2240.

09BD066 09BD06609BD066

09BD129 09BD129 09BD129

HeLa cells

Day 9

Day 17

PARP1 (pBD1678) DAPIEGFP

08bd085 08bd085 08bd085

Control (pBD1633) DAPIEGFP

pEBVsiPARP1-CAG2eGFP

Double KDSimple KD Simple KD

There is no limitation in the choice of the targeted gene.