Research Paper
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Acta Biochim Biophys Sin
2005,37: 779-783 |
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doi:10.1111/j.1745-7270.2005.00107.x |
Knockdown of Human p53
Gene Expression in 293-T Cells by Retroviral Vector-mediated Short Hairpin RNA
De-Long HAO#, Chang-Mei LIU#, Wen-Ji DONG, Huan GONG,
Xue-Song WU, De-Pei LIU*, and Chih-Chuan LIANG
National
Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100005, China
Received: July 12,
2005
Accepted: September
9, 2005
This work was
supported by the grants from the National High Technology Research and
Development Program of China (No. 2003AA216020 and No. 2002AA227012)
# These authors
contributed equally to this work
*Corresponding
author: Tel/Fax, 86-10-65105093; E-mail, [email protected]
Abstract RNA interference (RNAi)
is an evolutionarily conserved process of gene silencing in multiple organisms,
which has become a powerful tool for investigating gene function by reverse
genetics. Recently, many groups have reported to use synthesized oligonucleotides
or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection,
but this is still limited in its application, especially when it is necessary
to generate long-term gene silencing in vivo. To circumvent this
problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we
described two retroviral systems for delivering short hairpin RNA (shRNA)
transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can
substantially downregulate the expression of human p53 in 293-T cells.
Furthermore, the retroviral vector-mediated RNAi in our transduction system can
stably inactivate the p53 gene for a long time. Compared to
shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced
the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven
shRNAs were almost identical. The results indicate that retroviral
vector-delivered RNAi would be a useful tool in functional genomics and gene
therapy.
Key words retroviral vector; RNA interference; p53
RNA interference (RNAi) was initially discovered in Caenorhabditis
elegans, which is an evolutionarily conserved mechanism of
sequence-specific post-transcriptional gene silencing mediated by
double-stranded RNA (dsRNA) molecules that match the sequence of the target
gene [1]. Long dsRNA can be processed into short interfering RNA (siRNA) of 21-23 nucleotides
(nt) by Dicer, an RNase III-type endonuclease. Upon binding to the RNA-induced
silencing complex, double-stranded siRNA is unwound and targeted to homologous
mRNA, resulting in sequence-specific cleavage and degradation of mRNA [2-4]. RNAi has
been successfully used in plants and invertebrates for genetic analysis.
Double-stranded RNA longer than 30 nt may trigger interferon response by the
activation of the dsRNA-dependent protein kinase (PKR), leading to global
inhibition of protein synthesis in mammalian cells. Nonetheless, the
production of siRNAs that bind to specific endogenous mRNAs and induce their
degradation is now recognized as an ancient, evolutionarily conserved mechanism
that is widely employed in eukaryotic cells to inhibit protein production at
the post-transcriptional level [5]. siRNA of 21-23 nt synthesized in
vitro or expressed by DNA vector may bypass interferon response and induce
sequence-specific gene silencing. These approaches open the avenue of applying
the RNAi technique in gene function study and therapeutic research in mammalian
cells [6]. Many reports have described various DNA vector-based systems
expressing siRNAs in mammalian cells. In general, these DNA vectors contain an
RNA polymerase III promoter to express short dsRNA containing an inverted
repeat sequence in the form of a hairpin (or stem-loop) structure.
Short hairpin RNA (shRNA) was shown to be efficiently processed into
siRNA inside the cells, which resulted in sequence-specific gene silencing.
However, the delivery system that relies on transfection and the cell types
available for study limit the use of these DNA vectors. Recently, gene
silencing mediated by RNAi expressed in various viral vectors, including
retroviral and lentiviral vectors, was described [7-12]. Thus, viral vectors
combined with RNAi should provide useful tools to elucidate gene function by
the analysis of loss-of-function phenotype, and to explore the application of
RNAi in gene therapy. Furthermore, the use of retroviral vectors can greatly
expand the cell types available for RNAi analysis.
The tumor suppressor p53 is a sequence-specific transcription factor
that mediates many downstream effects such as growth arrest and apoptosis by
activation or repression of its target gene. The p53 protein has a short
half-life, but it can be stabilized by either point mutation of the gene or
interaction with specific DNA tumor virus factor, such as SV40 large T antigen
[13,14].
We chose p53 as the targeted gene to evaluate the effects of
RNAi with the two retroviral vector expression systems, pXSNhH1sip53-2 and
pXRNhH1sip53-2, for shRNA expression.
Materials and Methods
Cell culture
The GP-293 pantropic packaging cell line (Clontech, Palo Alto, USA) and 293-T cell line were maintained in Dulbecco's
modified Eagle's medium (DMEM; Gibco BRL, Carsbad, USA) supplemented with 10%
fetal bovine serum (FBS; Hyclone, Logan, USA) and antibiotics (100 mg/ml
streptomycin and 100 U/ml penicillin) at 37 ºC with 5% CO2.
Construction of retroviral
vectors
The retroviral vectors pXSN and pXRN were derived from pLXSN and
pLXRN vectors (Clontech) by deleting the 260 bp NheI/XbaI
fragment of 3' long terminal repeat (LTR). The human H1 promoter (-315/+1) from
pSuper (a gift from Dr. Yong-Feng SHANG, Beijing University, Beijing, China)
digested with BamHI/XhoI was cloned into pXSN and pXRN
upstream of either the SV40 early promoter (PSV40e)
or Rous sarcoma virus (RSV) promoter (PRSV) to
construct pXSNhH1 or pXRNhH1. Two synthetic oligonucleotides (5'-GATCCCCGACTCCAGTGGTAAT-CTACttcaagagaGTAGATTACCACTGGAGTC-TTTTTGGAAA-3'
and 5'-AGCTTTTCCAAAAAGACT-CCAGTGGTAATCTACtctcttgaaGTAGATTACCACT-GGAGTCGGG-3')
were annealed and ligated downstream of the H1 promoter in pXSNhH1 or pXRNhH1
to construct pXSNhH1sip53-2 or pXRNhH1sip53-2. The configuration of the
constructs pXSNhH1sip53-2 and pXRNhH1sip53-2 were verified by DNA sequencing.
Construction of recombinant
retrovirus and virus transduction into 293-T cells
The GP-293 packaging cells in 100 mm dishes were cotransfected using
Lipofectamine 2000 (Invitrogen, Carlsbad, USA) with 5-8 mg pVSV-G (Clontech) and 15-20 mg recombinant retroviral vector
(pXSNhH1, pXRNhH1, pXSNhH1sip53-2 or pXRNhH1sip53-2) for 24 h. After
transfection, the cells were incubated at 32 ºC to increase viral titer.
Forty-eight hours later, the supernatant containing the retroviral particles
was collected, filtered through the 0.45 mm low protein binding
syringe filter, and used to infect target cells.
293-T cells maintained
in DMEM were plated into six-well plates at 3´105 cells/well. Twenty-four hours later, the cells were infected with
viral supernatants in the presence of polybrene (5-8 mg/ml final concentration)
for 12 h, then added fresh medium with fresh viral supernatants. Twenty-four
hours later, the cells were incubated with fresh viral supernatant for
additional 12 h. The transfected 293-T cells were subcultured at an appropriate
density in fresh DMEM containing 1 mg/ml G418 (geneticin; Gibco BRL).
G418-resistant cell pools were readily established within a period of 7-10 d.
Western blot analysis
Infected 293-T cells were harvested at the indicated time points,
washed twice with cold phosphate-buffered saline, lysed in TNT lysis buffer (10
mM Tris, 150 mM NaCl, 1% NP-40, 10 mM NaF, 2 mM EDTA, 100 mg/ml 1,4-dithiothreitol,
100 mg/ml phenylmethylsulphonyl fluoride, 1 mg/ml aprotinin) for 30 min
on ice, and centrifuged at 15,000 g for 15 min to remove insoluble
materials. Protein concentrations were determined by BCA assay (Pierce,
Rockford, USA). Forty micrograms of lysate supernatant was separated using 12%
sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to
an Immobilon-P membrane (Millipore, Bedford, USA). The membrane was incubated
with anti-p53 (it reacts specifically with both wild and mutated human p53
protein) or anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, USA)
followed by incubation with horseradish peroxidase-conjugated goat anti-mouse
secondary antibody (Santa Cruz Biotechnology). Western blots were developed
using Western blotting luminal reagent (Santa Cruz Biotechnology).
Results and Discussion
To eliminate the interference of the 5' LTR counterpart on
virus replication [15], the retroviral vectors pLXSN and pLXRN were modified by
deleting the NheI/XbaI fragment of about 260 bp in 3' LTR
to generate pXSN and pXRN (Fig. 1).
We inserted the RNA polymerase III promoter of the human H1 small
nuclear RNA gene (PhH1) into the retroviral vectors pXSN and
pXRN. The H1 promoter was inserted into the multiple cloning sites of pXSN and
pXRN, with reverse orientation to the PSV40e- or PRSV-driven
neoR gene [Fig. 2(A)]. Subsequently, the synthesized inverted
repeats with an identical sequence to the human p53 gene was inserted
downstream of the H1 promoter, using 5 thymidines as the terminal signal [Fig.
2(B)]. The predicted hairpin RNA structure is shown in Fig. 2(C).
DNA sequencing demonstrated that the configurations of both the pXSNhH1sip53-2
and pXRNhH1sip53-2 constructs were correct.
The tumor suppression gene p53 is important in the regulation
of the cell cycle, and it also plays a crucial role in the progression of
cancer, as evidenced by the inactivation or loss of p53 in the majority of
human tumors. Because the 293-T cell line contains a high level of endogenous
wild-type p53, we used it as a model to test whether the retrovirus-mediated
RNAi could knockdown the expression of endogenous p53. The recombinant
retroviruses were generated by cotransfection of GP-293 cells with the envelope
plasmid pVSV-G, which confers the virus with pantropism, and the retroviral
vectors, pXSNhH1, pXRNhH1, pXSNhH1sip53-2 or pXRNhH1sip53-2. G418-resistant
293-T cell pools were established after infection with the recombinant
retrovirus.
Western blot analysis demonstrated that the expression levels of p53
were reduced dramatically compared with those of the internal control GAPDH [Fig.
3(A)], which suggested that retrovirus-delivered shRNA could efficiently
trigger the downregulation of p53 gene expression in a sequence-specific
manner in 293-T cells. The results also demonstrated that there was no obvious
difference in the inhibitory efficiency of the two systems [Fig. 3(B)].
Previously, we inhibited the human p53 gene expression with
shRNA expressed from human U6 promoter in the pXSN retroviral system [8]. In
the present study, H1 promoter was used to express shRNA to inhibit p53
gene expression in the pXSN and pXRN retroviral system, and the pXRN retrovirus
system had the similar efficiency as the pXSN system. In mammalian cells,
retroviral vector-mediated RNAi can be further applied to functional genomics,
so that a group of related individual genes can be silenced simultaneously and
their synergic functions can be systematically assessed. Lentiviral
vector-mediated transgenic knockdown suggested that RNAi might provide an
alternative approach to homologous gene targeting to create gene-knockout mice
[16,17]. Vectors based on lentiviruses are now in phase I clinical studies, and
there will be new applications for their use in the near future. In addition,
viral vector-mediated RNAi holds promises in gene therapy for cancers and
infectious diseases because it can result in loss-of-function phenotypes of
disease-related genes [18-20].
In conclusion, to facilitate stable and long-term knockdown in
cells, which are refractory to transfection-based gene transfer techniques, we
designed a retroviral vector system that permits delivery of stem-loop
cassettes. We reported the development of a versatile system of
retrovirus-based vectors, which makes it possible to achieve durable, high
efficiency siRNA-dependent gene silencing in a wide variety of cell lines. The
development of appropriate siRNA delivery vectors eventually may have important
applications, especially in gene therapy and genomic research. Although they
are not the perfect vectors for every purpose, for certain disease conditions
they are superior. Viral vectors combined with RNAi may provide useful tools to
elucidate gene function through the analysis of loss-of-function phenotypes.
Acknowledgements
We thank Dr. Yong-Feng SHANG (Beijing University, Beijing, China)
for providing the plasmid pSuper/H1, and Dr. Cheng-Han HUANG (The Lindsley F.
Kimball Research Institute, New York Blood Center, New York, USA) for
suggestions on the manuscript.
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