A P P L I C AT I O N N O T E

Lentiviral shRNAmirfor stable andregulatable RNAi

Product description
RNA interference (RNAi) has revolutionized the study of biology
and offers numerous applications in basic biology as wellas in drug discovery research. Since the discovery of RNAi,several tools have been developed to enable loss-of-function
studies in mammalian systems. Vector-based short hairpin RNA(shRNA) expression and synthetic short interfering RNA (siRNA)are currently the basis for all mammalian RNAi experiments(Figure 1). Each technology has specific advantages. shRNA
can be used to perform many of the experiments similar to
those done with siRNA. Additionally, when combined with viraldelivery and integration into the genome, shRNA has the added
benefit of long-term knockdown from a single treatment andthe incorporation of a genetic tag (or barcode) allowing highly
parallel pooled screening.
We have further refined this technology with the creation of
genome-scale microRNA-adapted shRNA libraries. shRNAmir
are expressed as primary-miRNA (pri-miRNA) transcripts.
These constructs were created by redesigning the well-studied
human miRNA, miR-30, to express an artificial siRNA/miRNA.
The stem of the primary miR-30 transcript was replaced withgene-specific duplexes for different target genes (Figure 2).
This design does not affect normal miRNA maturation andallows endogenous miRNA processing to produce maturesiRNAs. The shRNAmir design harnesses endogenous enzymatic
processing by the RNase III Drosha, which increases subsequent
Dicer recognition and specificity. shRNAmir triggers enter the RNAi
pathway ahead of either shRNA or siRNA, and are processed byboth Drosha and Dicer, leading to more siRNAs produced in thecell that are available for incorporation into the RISC complex fortarget mRNA degradation (Figure 3). [Silva et al. 2005, Boden et al.
2004, Cleary et al. 2004, Gregory et al. 2005].

Lentiviral shRNAmir for stable and regulatable RNAiAB

Figure 1: Evolution of RNAi triggers broadens RNAi applications.

Figure 2: shRNAmir design is based on the primary microRNA-30 (mir-30) transcript. A. Endogenous mir-30 primary transcript. B. shRNAmir
expressed from a miR-30 context. The mature mir-30 sequence has been replaced with a gene specific duplex. This design allows specificprocessing by Drosha and Dicer and active loading into the RISC complex.

Additionally, the lentiviral vector format can transduce

This tech note summarizes data provided by Rigel

hard‑to-transfect cell lines like primary and non-dividing.

Therapeutics from their pilot experiments conducted toThese lentiviral vectors in combination with shRNAmir design

evaluate the GIPZ and TRIPZ Lentiviral shRNA Genome Scaleoffer a powerful tool for RNAi studies.

Libraries. Seventy-eight GIPZ shRNAmir constructs designed

Whole genome shRNAmir lentiviral libraries have beencreated using both the GIPZ™ lentiviral vector (Figure 4)
that incorporates green fluorescent protein (GFP) to track
shRNAmir expression, and the TRIPZ™ lentiviral vector(Figure 5), which shares many of the benefits of GIPZ andincludes a tetracycline (doxycycline) inducible promoter that
produces tightly regulatable RNAi.

against 19 target genes were transduced into multiple celllines at low multiplicity of infection (MOI). The GIPZ shRNAmirexerted knockdown effects at the both RNA and protein
level. Knockdown of the targeted genes with GIPZ shRNAmir
mimicked known cellular phenotypes induced by small
molecule inhibitors. The GIPZ shRNAmir were also employed
to dissect the components in a signaling pathway using aSMAD reporter system. Their findings support previous data
showing that the shRNAmir constructs produced effective
knockdown at low copy.

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Lentiviral shRNAmir for stable and regulatable RNAi

RRE

TRE

UBC

tRFP

rtTA3

IRES

PuroR

WPRE

shRNA
3' SIN LTR

5' LTRpTRIPZ
AmpRA

pUC ori

SV40 ori

BC

hCMV
tGFP

IRES

PuroR

shRNA

WPREΨ

RRE

5' LTRDpSMART 2.0

500 ng/ml Dox 48 hr

500 ng/ml Dox 72 hr

EAmpR

Post Dox 24 hr

Figure 3: shRNAmir is processed via the endogenous microRNApathway. 1) shRNAmir triggers modeled on primary miRNA
are processed by Drosha and Dicer to produce mature siRNAtargeting a complementary mRNA. 2) shRNA triggers areapproximately modeled on precursor miRNA and are processedby Dicer to produce mature siRNA. 3) Chemically synthesizedsiRNA enter the RNAi pathway post-Dicer cleavage andincorporate into RISC to target complementary mRNA. All three
RNAi triggers use the endogenous RNAi pathway but have distinct
entry points.

3' SIN LTR

Non Silencing shRNAmir
No Doxycycline (Dox) 48 hr

pUC ori

SV40 ori

Post Dox 72 hr

U6hPGK

Figure 5: A. The TRIPZ vector incorporates many of the sameelements of the GIPZ lentiviral backbone including a fluorescentmarker (tRFP) of shRNAmir expression. Additionally, it contains
all of the components necessary for inducible expression ofthe shRNAmir. B-D. tRFP expression is shown as a marker ofcontrolled, inducible expression from this vector. Transducedcells were fed media containing 500 ng/mL doxycycline (Dox) andmaximal induction was seen within 72 hours. E. Subsequently, Thecells were washed and fed medium without doxycycline. Seventytwo hours later tRFP reflected a minimal level of expression.

hCMV
tGFP

IRES

PuroR

WPRE

ΨRRE

shRNA
3' SIN LTR

5' LTR

pGIPZ
AmpR

pUC ori

AmRNA expression was measured by qPCR. H1299 humanB
tRFP

rtTA3lung carcinoma cells were transduced at an MOI of 1-2,IRES

PuroR

selected with puromycin for 48 hours and gene expression

WPRE

was measured by qPCR. Three independent experiments

shRNApTRIPZ

3' SINLTRΨRRE

UBC

Figure 4: The pGIPZ vector expresses turboGFP (tGFP) as amarker of shRNAmir expression. A. Vector map highlighting thecomponents of pGIPZ. shRNAmir is constitutively expressed asa polycistronic vector expressing tGFP, puromycin resistancemarker (Puror) and the shRNAmir. B. HEK293T cells transduced
by GIPZ at a low MOI. Green fluorescence is an indicator that theshRNAmir-containing transcript is being expressed.

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Seventy-eight shRNAmir in either the GIPZ or the TRIPZlentiviral vectors were evaluated and percent remaining

SV40 ori

TRE

Gene expression knockdown

were performed for each shRNAmir. Previous work done inconcert with the National Cancer Institute showed that theGIPZ library was capable of achieving ≥ 70% knockdown inapproximately 2 of 3 hairpins. The findings here were similar.
In H1299, 72% of the shRNA were shown to knockdown
gene expression by ≥ 70% (Figure 6). Overall this validated
the previous expectations of the library for consistency andlevel of knockdown.

3Lentiviral shRNAmir for stable and regulatable RNAiA

100%

Revvity GIPZ

100%

B

95%

90%
80%
70%

70%

64%

61%

60%

54%

50%

50%

45%

40%

40%

23%

20%
10%

Revvity TRIPZ

80%

60%

30%

100%

90%

84%

80%

100%

16%
6% 7%

28%

25%
22%
20%
14%14%

30%

22%22%

20%

9% 10%

8%

35%

35%

23%25%22%24%
17%

24% 26%

10%

1%
0%

0%

Figure 6: Demonstration of knockdown efficiency. In H1299, a human lung carcinoma cell line, 72% of the shRNA were shown to knock down
gene expression by ≥ 70%. The red line denotes this level of knockdown. Multiple hairpins target each gene either in A. GIPZ or B. TRIPZ.
All samples were measured using qPCR. The value shown is an average of three independent experiments.

Constitutive and inducible knockdown
at the protein level
mRNA knockdown is a quick and useful validation ofhas been added to induce expression of the shRNAmir.

shRNAmir function. However, the resulting decrease in

These protein levels should be the same if there is noprotein is usually what is thought to alter phenotypes.

unwanted expression of the shRNAmir, and this is what was

The cell surface marker AXL was targeted in HeLa

found by using a FACS assay and measuring AXL (Figure 7).

cells and changes in protein level were measured bya fluorescent immunocytochemistry assay using FACS

Untransduced - ‘OFF’ (-DOX) = No Leak
‘OFF’ (-DOX) - ‘ON’ (+DOX) = Inducible Knockdown

analysis to determine the ability of GIPZ and TRIPZ
100to knockdown protein levels. Every shRNAmir in bothvectors provided > 70% knockdown of the RNA in HeLa
expression provides experimental flexibility, and theuninduced state is an ideal control for many experiments.
However, one concern about inducible expression systems
is the possibility of a basal expression level in the absenceof the inducing agent (tetracycline in this case) which is% of Max.

cells (data not shown). The option of inducible shRNAmir

80‘ON’ (+DOX)‘OFF’ (-DOX)

60

Untransduced
Unstained

40
20

commonly referred to as ‘leaky’ expression.
There are two main components on the pTRIPZ vector
enabling induction: the tetracycline response element (TRE)

0100

101

102

FL 8 Log: APC

and the transactivator. The transactivator expressed frompTRIPZ is rtTA3, a modified form optimized for increased
response to tetracycline without increasing background.
TRIPZ demonstrated tightly controlled expression. The levelof protein expression is compared between untransduced
cells and cells that are transduced but no tetracycline (‘OFF’)

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Figure 7: Decrease in protein level without ‘leaky’ expression
shown by FACS analysis. Immunocytochemistry was used todetect the AXL protein by FACS. The leftshift of the peaks
denotes a decrease in the AXL protein level after knockdownwith TRIPZ shRNA targeting AXL. The untransduced and ‘OFF’sample primarily overlap suggesting very little uninduced or leaky
expression. The left-shifted red peak compared to the blue shows
the knockdown.
4

Lentiviral shRNAmir for stable and regulatable RNAi

Phenotypic validation
A high content screen ultimately relies on phenotypicchanges. In this example, H1299 cells were transducedwith constructs from the GIPZ library targeting genes
associated with cell cycle progression and cancer.
Many of the phenotypes presented as polyploidy including
endoreduplication, a cellular state in which the genomeof the cell is duplicated but it does not under go mitosis.
The polyploidy phenotype can present as one very large
nucleus or multinucleated phenotypes and matched very
well with previous data using siRNA or small molecule
described in the literature (Figure 8, see page 4).
The library was able to mimic known cellular phenotypes
induced by small molecule inhibitors for every target
gene chosen, thereby validating its use as a screening toolwith a phenotypic assay. Any of these results could beincorporated as a positive control into a high content screen
for additional novel genes inducing the phenotype.

Figure 8: Knockdown of the targeted genes produces phenotypes
known to result from disrupting the cell cycle. AURKB, andPLK1 knockdown produced an endoreduplication phenotype.
EG5 knockdown produced the expected preprometaphase‘stellate’ phenotype and endoreduplication. Knockdown of ACTA1
produced a multinucleated phenotype.

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Figure 9: The cell line HeLa/ SMAD-gRL was engineered toexpress luciferase when stimulated with TGF-β. The cell line
allowed luciferase to be used as a readout for signaling throughthis pathway. Unknown genes’ contribution to this pathway
can now be correlated to luciferase activity. Genes without
redundancy in the pathway will result in a stronger disruption
in luciferase activity when knocked down. The converse is also
true. This is reflected in the luciferase readout of TGFbR1 whichis required for signaling and the SMADS that have redundant
partners with alternate downstream signaling. (Adapted from
Rigel’s data presentation.)5

Lentiviral shRNAmir for stable and regulatable RNAi

Pathway analysis
One of the more sophisticated experiments that can beluciferase from a SMAD response element-based promoter

done with RNAi is to dissect the components in a signaling(Figure 9) coupling TGF-β signaling activity to luciferase

pathway. Changes in many genes’ expression can be

activity, a functional readout more amenable to a highdifficult to detect directly. Using a reporter system canthroughput assay. Dose response curve was first generated

alleviate this by linking expression of a reporter protein

by stimulation of this pathway without disrupting gene(for example, luciferase) to a promoter that is known to beexpression (Figure 10a). After each gene suspected to be inactivated when the pathway of interest is involved. Thethe pathway was knocked down the resulting dose responsewell characterized TGF-β pathway was used as a model.

curve was compared back to the control (Figures 10 b-d).

TGF-β, a cell signaling molecule involved in proliferation andChanges in luciferase activity were used to determine thedifferentiation in many cell types regulates gene expression

relative contribution of many genes in the TGF-β/SMAD

through SMAD pathway. Signaling through this pathway

signaling pathway. Knockdown of the TGF-β receptor,

initiates with the TGF-β-receptor binding TGF-β and involves

TGFbR1, significantly disrupted luciferase expression

many elements of the SMAD signaling pathway. SMADs(Figure 10b). Knockdown of SMAD2 and SMAD4 (Figures 10crepresent a broad family of genes that can overlap in theirand 10d respectively) resulted in a significant but incomplete

contribution to signaling [reviewed in Derynk, R. et al. 2003].

disruption of the reporter. This reflects the redundant nature

A cell line (HeLa/SMAD-gRL) was designed to expressesof multiple SMADs with overlapping signaling roles.

AB

CDFigure 10: Luciferase as a read out for SMAD signaling using the cell line HeLa/SMAD-gRL. A. Control dose response curve was generated
from the HeLa/SMADgRL cell line without disrupting the pathway. SMAD genes known to participate in this signaling pathway were knocked
down using multiple hairpins to each gene (their clone IDs are shown in the legend). A decrease in the luciferase output suggests a disruption
in the SMAD signaling pathway. The response curves were compared to the control. Multiple hairpins to three of the target genes are shown
here B. TGFbR1, the TGF-β receptor, C. SMAD2 and D. SMAD4.

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Lentiviral shRNAmir for stable and regulatable RNAiThis data provides benchmarks for luciferase activity while

1. One of the main concerns of an RNAi experiment is thedisrupting genes known to be involved in the pathway.

possibility for off-target effects. The ability to achieve

Going forward, using the current data to gauge participation

significant knockdown at low MOIs will reduce thein TGF-β signaling pathway, this assay is amenable to a highpossibility of concentration dependent off-target effects.

throughput screen of the entire genome. Knockdown of theTGF-β receptor, TGFbR1, significantly disrupted luciferase
expression (Figure 10b). Knockdown of SMAD2 and SMAD4(Figures 10c and 10d respectively) resulted in a significant

2. Fewer resources can be used to perform more experiments.
3. Any downstream event dependant on knockdown will bemore robust. For example, this was shown for the TGF-β

but incomplete disruption of the reporter. This reflects

pathway (Figures 9 and 10). Known phenotypes caused by

the redundant nature of multiple SMADs with overlapping

small molecule inhibitors were mimicked and the essential

signaling roles. This data provides benchmarks for luciferase

components of the pathway were able to be distinguishedactivity while disrupting genes known to be involved in thefrom the redundant components.

pathway. Going forward, using the current data to gauge
participation in TGF-β signaling pathway, this assay isamenable to a high throughput screen of the entire genome.

References
1. Silva, J.M. et al. (2005) Second-generation shRNA libraries

Conclusion

covering the mouse and human genomes. Nat. Genet. 37,

The ability of the GIPZ and TRIPZ lentiviral shRNAmir
constructs to produce effective knockdown has been

1281–1288.
2. Boden, D. et al. (2004) Enhanced gene silencing of HIV-1
specific siRNA using microRNA designed hairpins.

evaluated at the mRNA and protein level as well as in

Nucleic Acids Res. 32, 1154–1158.

functional analysis. qPCR results confirmed not only thata majority of the constructs in this library are capable of

3. Cleary, M.A. et al. (2004) Production of complex nucleic

inducing sufficient knockdown (~ 2 out of 3), but that this canacid libraries using highly parallel in situ oligonucleotide

be achieved at low MOIs in most cases. This has significant

synthesis. Nat Methods 1, 241–248.

advantages for the customer who is interested in knockdown

4. Gregory, R.I. et al. (2005) Human RISC couples microRNAof a single gene or the customer who is interested in a larger

biogenesis and posttranscriptional gene silencing.

scale screening tool:

Cell 123, 631–640.
5. Derynk, R. et al. (2003) Smad-dependent and Smadindependent pathways in TGF-beta family signalling.
Nature 425, 577-84.

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