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Introduction
RNA interference (RNAi) is a powerful tool for inhibiting
gene expression in a wide variety of organisms. When
double-stranded RNA is introduced into cells, the
ribonuclease III Dicer processes the double-stranded RNA into
small fragments of about 21 nucleotides in length (termed
siRNAs) that trigger the RNAi
mechanism[1-3]. Then, the siRNAs are incorporated into a protein complex, known as
the RNA-induced silencing complex (RISC), which in turn
unwinds the duplex siRNA in an ATP-dependent
manner[4]. After the unwinding, RISC uses the siRNA antisense strand
as a guide to specifically cleave the complementary mRNA,
which is further processed for
degradation[4-6].
There are different ways to induce mRNA degradation
using RNAi. Several methods for preparing siRNA have
been developed, such as chemical synthesis, in
vitro transcription, siRNA expression vectors, and polymerase
chain reaction (PCR) expression cassettes. For example, short
hairpin RNAs (shRNAs) transcribed by RNA polymerase III
promoter-based vectors have been used as RNAi triggers in
a variety of cell lines[7,8]. These RNA polymerase III (RNA
Pol) vectors have a variety of advantages over the siRNAs.
First, they greatly reduce the cost of RNAi synthesis,
making RNAi a viable tool for screening the function of large
numbers of genes. Second, because hairpins are transcribed
inside cells, it is possible to establish inducible systems for
RNAi both in vitro and in
vivo[9-11]. Third, it is also possible
to generate knock-down phenotypes to resemble knock-out
animals without affecting the germline, as long as this
strategy is combined with inducible
systems[12-14]. Therefore, it is relatively easy to establish cell cultures using retroviral or
adenoviral vectors that stably express the hairpin for
RNAi[10,15-18].
Although there has been much progress in the RNAi
field, it is not completely understood why some sequences
are refractory to RNAi. Some groups have investigated this
lack of activity, but at present, choosing the most effective
siRNA sequence to knock down an mRNA is still a
trial-and-error task. Thus, it is generally accepted that several
sequences must be designed to achieve the highest RNAi
efficiency, making the silencing experiments costly and slow.
It would be advantageous to have a more efficient method
for the production of a wide variety of sequences in a simple
step, instead of using long DNA oligonucleotides or several
PCR step-specific methods[8,19-21].
Here we describe an inexpensive, easy to implement and
automated method to produce DNA cassettes for RNAi
experiments using a single oligonucleotide. With this method
hundreds of DNA cassettes can be synthesized in a 1-step
reaction. This method not only reduces the cost of testing
sequences for RNAi experiments, but also accelerates the
exploration of multiple-gene sequences.
Materials and methods
Construction of DNA cassettes and plasmid vectors
To construct the plasmid vector pBB4H1, the human H1
promoter was PCR amplified from human genomic DNA using
the following primers: forward 5¡¯-CCATGGAATTCGAA-CGCTGACGT-3¡¯ and reverse
5¡¯-GCAAGCTTTGGTCTCA-TAAGAACTTATAAGATTCCC-3¡¯, which contain one
EcoRI and one HindIII restriction site, respectively. The PCR
product was subcloned in the pDRIVE vector (Qiagen, Valencia,
CA) following the manufacturer¡¯s instructions. The
pDRIVE-H1 vector was then digested with
EcoRI and HindIII to obtain a DNA fragment of ~230 base pairs, which contained
the H1 promoter. This fragment was then cloned into the
EcoRI and HindIII site of the pBlueBac4 vector (Invitrogen,
Carlsbad, CA) to obtain the pBB4H1 vector.
To generate the DNA cassettes the following chemically
synthesized oligonucleotides were used: hpGFP1
5¡¯-GC-AAGCTTCCCCAAAAACCACTACCTGAGCACCCA
GGGG-CCCC-3¡¯, hpGFP2
5¡¯-GCAAGCTTCCCCAAAAAGGGCGA-GGAGCTGTTCACCGG
GGCCCC-3¡¯, hpGFP3 5¡¯-GCAAGC-TTCCCCAAAAACGGCCACCAGTTCAGCGT
GGGGCCCC-3¡¯, hpGFP4
5¡¯-GCAAGCTTCCCCAAAAAGGAGGACGGC-AACATCCT
GGGGCCCC-3¡¯, hpTRPC4 5¡¯-GCAAGCTT-CCCCAAAAAUUACUCGUCAACAGGCGGACGG
GGCC-CC-3¡¯. Note that the sequence upstream of the 5 A¡¯s
contain-ed the HindIII restriction site (underlined), followed by
4 C¡¯s, and was included in the oligonucleotide design
because it allows easy cloning of the DNA cassette and allows
the RNA pol III to initiate in the first of the 5 A¡¯s, exactly 30
nucleotides after the TATA box. The sequence in italics
corresponds to the eGFP mRNA.
To construct the DNA cassettes, 10 μL of each
oligonucleotide (~35 μmol/L) were heated for 5 min to 94 °C.
When the temperature reached 40 °C, 10 μL of a reaction mix
was added [reaction mix: 2 mmol/L dNTPs; 5 U Klenow
enzyme (Invitrogen); 2 μL 10×reaction buffer
2 (Invitrogen); 10% Me2SO;
H2O to 10 μL]. All the reactions were performed
identically using a thermo-cycler to precisely control the
temperatures (Figure 1).
Then, the Klenow products were heated to 65 °C to
inactivate the enzyme, digested with HindIII and cloned into the
HindIII restriction site of the pBB4H 1 vector previously
dephosphorylated with alkaline phosphatase (Roche, Basel
Switzerland). Only the correct cassettes would be ligated
into the HindIII cohesive ends. We did not observe clones
with tandem-repeated cassettes.
The sequences targeting the eGFP mRNA were chosen
based on 2 criteria: (i) because they had fewer than 3 G or C
repeats to prevent G-quartet formation; and (ii) because they
were distributed along the eGFP mRNA.
Cell culture and transfection HeLa cells were grown in
Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) containing
10% fetal bovine serum and antibiotics, and were seeded
every 3 d following standard procedures at 37 °C. The day
before transfection, cells were grown without antibiotics in
12 well plates. Vectors containing the DNA cassette were
transfected using lipofectamine 2000 (Invitrogen) at
different concentrations following the manufacturer¡¯s instructions.
The day after transfection, the cells were washed once with
phosphate-buffered saline (PBS) and treated with 5 MOI of a
recombinant adenovirus encoding the green fluorescent
protein (eGFP) gene under the transcriptional control of the
cytomegalovirus (CMV) promoter[22]. Four hours later, the cells
were washed with PBS and the media was replaced.
Twenty-four hours later the fluorescence was evaluated using a
FACScan flow cytometer (Becton & Dickinson, Franklin
Lakes, NJ). To evaluate the fluorescence intensity, 10 000
events were counted in at least 3 independent experiments
for each concentration.
Confocal microscopy The HeLa cells that were treated
with the plasmids and adenovirus as described were washed
with PBS, trypsinized and plated on sterile coverslips.
Twenty-four hours later, the cells were analyzed with a
confocal microscope (Bio-Rad, Hercules, CA) using a 40×
objective. The confocal images were analyzed using the
Confocal Assistant freeware software.
Results
Construction of DNA cassettes For the construction of
the DNA sequence that served as a template for shRNA
transcription, we followed the principle of DNA
amplification commonly used in PCR reactions (Figure 2A). That is,
any given sequence that serves as a primer could be
amplified at its 3¡¯ end with a proper DNA polymerase. In RNAi
hairpins, the 2 strands that form the stem are fully
comple-mentary, so it is possible to use only a single
oligonucleotide that is self-complementary within its 3¡¯ end (Figure 2B).
After the self-complementary structure is formed, it is
possible to further amplify the structure so that the
oligonucleo-tide becomes completely double-stranded DNA.
The self-complementary pairing must be strong enough to allow the DNA polymerase to initiate the reaction at the
selected temperature. We chose the sequence
5¡¯-GGGGCCCC-3¡¯ due to its high Tm
(37 °C) and because its palindrome nature permits the self-complementary structure. Furthermore,
this sequence allows amplification using the large (Klenow)
fragment of the DNA polymerase, whose working
temperature is 37 °C. Thus, because the 5¡¯-GGGGCCCC-3¡¯ sequence
is amplified and lies between the sense and antisense strand,
it also functions as a loop for the shRNA (Figure 4).
Finally, most RNAi vectors contain an RNA pol III
promoter such as U6 or H1, which is used to initiate the
transcription of the shRNA. When the RNA pol III finds 5
consecutive T¡¯s, it terminates the transcription and removes the
last three nucleotides on the 3¡¯ end[17,20]. Therefore, we included 5 A¡¯s in the 5¡¯ end in the oligonucleotide; when the
Klenow fragment synthesizes the antisense strand, it
generated the 5 T¡¯s, which will be needed to terminate the
transcription by RNA pol III inside the cell (Figure 2C). The
expected shRNA structures that will form inside the cells are
shown in Figure 4. All these structures were modeled using
the Mfold server (see Materials and methods).
To determine if it was possible to construct the DNA
cassette for shRNA vectors with this method, we designed
and tested its ability to amplify different oligonucleotides.
These oligonucleotides were prepared to target sequences
from eGFP and a control oligonucletide containing an
eGFP-unrelated sequence. eGFP oligonucleotide sequences were
selected to have low GC repeats to prevent G-quartet
forma-tion. Figure 3 illustrates the result of a typical filling-in
reaction, showing that the Klenow fragment produced
double-stranded DNA from single oligonucleotides.
Therefore, the self-complementary 5¡¯-GGGGCCCC-3¡¯
sequ-ence is a good template for DNA amplification. Furthermore,
all cassettes were constructed simultaneously in a single
step reaction, so the 1-oligonucleotide method could be
automated to perform a large number of reactions in a short
period of time (see Materials and methods and Figure 1). We
cloned the DNA cassettes in the vector under the
transcriptional control of an RNA pol III (H1) promoter, and all
cassettes were sequenced to confirm their integrity.
Efficacy of the 1-oligonucleotide method in RNAi
experiments To test the ability of the DNA cassettes to trigger
RNAi activity, HeLa cells were transfected with the plasmid
vectors containing the cassettes targeting the mRNA from
eGFP and a control construct. The day after transfection,
cells were incubated with a recombinant adenovirus
encoding the eGFP, and 24 h later the cell fluorescence was
evaluated. All the tested sequences silenced eGFP
expression and, as expected, there was no gene silencing with the
under the transcriptional control of the strong CMV
promoter in the adenoviral vector, it is still possible to induce
gene silencing using the cassettes described here.
Previously reported methods to produce RNAi using
DNA cassettes to generate shRNAs in mammalian cells
require long oligonucleotides or several PCR reactions that
are sequence and step
specific[17,20,21,24-28]. For example, in
the method reported by Gou et al, at least three
oligonucleotides (1 forward and 2 reverse) are used in 2 sequential PCR
reactions to generate the DNA
cassette[21]. Once the 2 PCR reactions conclude, PCR products require further cleaning
before transfection into cells. Another method previously
reported is based on primer extension, and the DNA
cassettes are cleaned and introduced in the cells
directly[26]. Although the latter method uses only 2 oligonucleotides to
construct each DNA cassette, these oligonucleotides are
fairly long (approximately 100 nucleotides), considerably
increasing the cost of constructing multiple cassettes.
There are a number of effective methods that have been
designed over the past few years to produce RNAi in
mammalian cells[29], but unfortunately none of these methods is
easily automated because they require many step-specific
reactions. Using the method described here, we
simultaneously produced a large number of different DNA cassettes
targeting different mRNAs (unpublished data). This is
possible because the process can be automated to perform many
different reactions using the same temperature, time and
reactant conditions using a thermocycler.
The advantages of the method described here over
previously reported methods are: (i) a single oligonucleotide is
sufficient to generate a DNA cassette for shRNA production,
significantly reducing the price of testing multiple sequences;
(ii) oligonucleotides for cassette construction are easy to
design: a 21-25 nucleotide sense target sequence is flanked
by 5¡¯-AAAAA- and -GGGGCCCC-3¡¯, thus the length of the
oligonucleotide is considerably shorter; and (iii) the method
is automated, allowing the construction of a wide variety of
DNA cassettes in one step in a few minutes.
Recently it was shown that it is also possible to express
synthetic miRNAs using RNA pol II promoters, which
allows still more control over stem-loop RNA expression,
because many RNA pol II promoters function in a
inducible/tissue-specific fashion[30]. We are currently constructing
DNA cassettes by using the 1-oligonucleotide method for
use with RNA pol II promoters (unpublished results).
Because this type of polymerase does not terminate the
transcription after 5 consecutive thymidines it is not necessary
to include 5 adenines in the oligonucleotide, which further
reduces the length and price of the DNA cassette.
transfection of the unrelated construct (Figure 4). A
dose-response experiment showed that the most effective plasmid
concentration to achieve RNAi was 1-3.75 μg/mL. Of 4 tested
DNA cassette sequences (termed hpGFP1 to 4), hpGFP 2, 3
and 4 inhibited eGFP expression/fluorescence by more than
70%. The less efficient cassette (hpGFP1) inhibited eGFP
expression by 50% at 1 μg/mL (Figure 5). The
EC50 of each shRNA confirms this result; the less effective shRNA is
hpGFP1, with an EC50 of approximately 0.5 μg/mL, whereas
hpGFP 2-4 have EC50 values of approximately 0.05 μg/mL
(Figure 5). The finding that not all DNA cassette sequences
had the same potency confirms the fact that a variety of
sequences must be designed for the same target in order to
find the most effective RNAi inducer. However, it was
surprising that the hpGFP 1 sequence was the least effective of
all tested sequences. This result was interesting because
this sequence had been shown to be a strong inhibitor of
eGFP expression in a previous study[23]. The dose-response
experiment with the unrelated cassette did not influence the
eGFP fluorescence (data not shown). Together, these
results suggest that diverse DNA cassettes must be produced
by the 1-oligonucleotide method in order to identify the best
sequence for RNAi.
Discussion
In the present work we developed a novel, easy and
efficient 1-oligonucleotide method to generate DNA cassettes
for RNAi vectors. To test the capability of the method, we
designed DNA cassettes to target the mRNA from eGFP. For
this purpose a recombinant adenovirus encoding the eGFP
gene was used to infect HeLa cells. Only cells infected with
the virus and transfected with specific anti-GFP cassettes
strongly inhibited eGFP fluorescence. The level of
inhibition achieved with this method resembles the inhibition when
RNAi is used to knock-down the expression of endogenous
genes, because transfections usually reached 60%-75% of
the cells, and the level of adenovirus infection almost reached
100%. The results obtained here suggest that eGFP
expression was strongly reduced in the cells that were transfected
with the DNA constructs, as illustrated in Figure 5. The fact
that hpGFP1 was less efficient at knocking down eGFP
expression suggests that a variety of RNA sequences are
needed in order to find the most effective. Although this
sequence has already been used by
others[23], it is reasonable that there could exist sequences that are even more
effective; this method is an easy way to construct a large
variety of DNA cassettes for 1 or more genes. Together,
these results demonstrate that, although eGFP expression is
In conclusion, we reported here a novel method for
constructing large libraries of RNAi cassettes quickly and in a
cost-effective manner, thus making the exploration of the
function of a large number of genes an easier task and
facilitating functional genomic studies.
Acknowledgements
We thank Agustin LUZ for providing genomic DNA for
cloning the H1 human promoter, and Erendira
AVENDAÑO and members of the laboratory for helpful discussions.
References
1 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi:
double-stranded RNA directs the ATP-dependent cleavage of mRNA at
21 to 23 nucleotide intervals. Cell 2000; 101: 25-33.
2 Hammond SM, Bernstein E, Beach D, Hannon GJ. An
RNA-directed nuclease mediates post-transcriptional gene silencing in
Drosophila cells. Nature 2000; 404: 293-6.
3 Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a
bidentate ribonuclease in the initiation step of RNA interference.
Nature 2001; 409: 363-6.
4 Nykanen A,Haley B, Zamore PD. ATP requirements and small
interfering RNA structure in the RNA interference pathway. Cell
2001; 107: 309-21.
5 Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song
JJ, et al. Argonaute2 is the catalytic engine of mammalian RNAi.
Science 2004; 305: 1437-41.
6 Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD.
Asymmetry in the assembly of the RNAi enzyme complex. Cell
2003; 115: 199-208.
7 Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS.
Short hairpin RNAs (shRNAs) induce sequence-specific silencing
in mammalian cells. Genes Dev 2002; 16: 948-58.
8 Brummelkamp TR, Bernards R, Agami R. A system for stable
expression of short interfering RNAs in mammalian cells.
Science 2002; 296: 550-3.
9 Alibu VP, Storm L, Haile S, Clayton C, Horn D. A doubly
inducible system for RNA interference and rapid RNAi plasmid
construction in Trypanosoma brucei. Mol Biochem Parasitol 2005;
139: 75-82.
10 Hosono T, Mizuguchi H, Katayama K, Xu ZL, Sakurai F,
Ishii-Watabe A, et al. Adenovirus vector-mediated
doxycycline-inducible RNA interference. Hum Gene Ther 2004; 15: 813-9.
11 Czauderna F, Santel A, Hinz M, Fechtner M, Durieux B, Fisch G,
et al. Inducible shRNA expression for application in a prostate
cancer mouse model. Nucleic Acids Res 2003; 31: e127.
12 Robinson R. RNAi therapeutics: how likely, how soon? PLoS
Biol 2004; 2: E28.
13 Wesley SV, Liu Q, Wielopolska A, Ellacott G, Smith N, Singh S,
et al. Custom knock-outs with hairpin RNA-mediated gene
silencing. Methods Mol Biol 2003; 236: 273-86.
14 van den Haute C, Eggermont K, Nuttin B, Debyser Z, Baekelandt
V. Lentiviral vector-mediated delivery of short hairpin RNA
results in persistent knockdown of gene expression in mouse brain. Hum Gene Ther 2003; 14: 1799-807.
15 Liu XD, Ma SM, Liu Y, Liu SZ, Sehon A. Short hairpin RNA and
retroviral vector-mediated silencing of p53 in mammalian cells.
Biochem Biophys Res Commun 2004; 324: 1173-8.
16 Zhao, LJ, Jian H, Zhu H. Specific gene inhibition by
adenovirus-mediated expression of small interfering RNA. Gene 2003; 316:
137-41.
17 Xia XG, Zhou H, Ding H, Affar el B, Shi Y, Xu Z. An enhanced
U6 promoter for synthesis of short hairpin RNA. Nucleic Acids
Res 2003; 31: e100.
18 Shen C, Buck AK, Liu X, Winkler M, Reske SN. Gene silencing
by adenovirus-delivered siRNA. FEBS Lett 2003; 539: 111-4.
19 Brummelkamp TR, Bernards R, Agami R. Stable suppression of
tumorigenicity by virus-mediated RNA interference. Cancer Cell
2002; 2: 243-7.
20 Paddison, PJ, Caudy AA, Hannon GJ. Stable suppression of gene
expression by RNAi in mammalian cells. Proc Natl Acad Sci USA
2002; 99: 1443-8.
21 Gou D, Jin N, Liu L. Gene silencing in mammalian cells by
PCR-based short hairpin RNA. FEBS Lett 2003; 548: 113-8.
22 Tattersfield AS, Croon RJ, Liu YW, Kells AP, Faull RL, Connor
B. Neurogenesis in the subventricular zone following transcranial
magnetic field stimulation and nigrostriatal lesions. J Neurosci
Res 2004; 78: 16-28.
23 Billy E, Brondani V, Zhang H, Muller U, Filipowicz W. Specific
interference with gene expression induced by long, double-stranded
RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl
Acad Sci USA 2001; 98: 14428-33.
24 Castanotto D, Rossi JJ. Construction and transfection of PCR
products expressing siRNAs or shRNAs in mammalian cells.
Methods Mol Biol 2004; 252: 509-14.
25 Castanotto, D, Li H, Rossi JJ. Functional siRNA expression from
transfected PCR products. RNA 2002; 8: 1454-60.
26 Sioud M, Leirdal M. Potential design rules and enzymatic
synthesis of siRNAs. Methods Mol Biol 2004; 252: 457-69.
27 Yu, JY, DeRuiter SL, Turner DL. RNA interference by
expression of short-interfering RNAs and hairpin RNAs in mammalian
cells. Proc Natl Acad Sci USA 2002; 99: 6047-52.
28 Paddison PJ, Caudy AA, Sachidanandam R, Hannon GJ. Short
hairpin activated gene silencing in mammalian cells. Methods
Mol Biol 2004; 265: 85-100.
29 Hannon, GJ, Conklin DS. RNA interference by short hairpin
RNAs expressed in vertebrate cells. Methods Mol Biol 2004;
257: 255-66.
30 Zeng Y, Cai X, Cullen BR. Use of RNA polymerase II to
transcribe artificial microRNAs. Methods Enzymol 2005; 392:
371-80.
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