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Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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specifically reduce surface expression of these coreceptors and their
respective mRNA in a thymoma cell line model. We show that RNAi
activity is only caused by a subset of siRNAs complementary to the mRNA
target and that ineffective siRNAs can compete with effective siRNAs.
Using primary differentiated T lymphocytes, we provide the first
evidence of siRNA-mediated RNAi gene silencing in normal nontransformed
somatic mammalian lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian RNAi was first described in mouse embryos using long dsRNA (11, 12). Then, following the analysis of the structure of the intermediate in this process, small interfering RNAs (siRNAs) were used to silence genes in mammalian tissue culture (13, 14). Most of the RNAi pathway genes discovered in plant and worm screens are also present in mouse and human sequence databases, supporting evidence that a conserved RNAi pathway exists in mammals. One of the more notable exceptions is the RNA-dependent RNA polymerase gene, which has been shown to be involved in the amplification of the dsRNA in Caenorhabditis elegans (15, 16). This might imply that perpetuation of the RNAi response in mammals differs from that of lower organisms.
Recent reports have demonstrated gene silencing by siRNA in mammalian cells (17, 18, 19, 20, 21, 22). However, despite these initial reports, many uncertainties remain concerning the mechanism, physiologic relevance, and ubiquity of RNAi in mammalian cells. Although studies in tumor cell lines have demonstrated siRNA-mediated RNAi, it remains a major question as to whether primary cells from fresh tissues can undergo the RNAi response. Furthermore, little is known about the efficiency and longevity of siRNA-mediated RNAi gene suppression. In this report, we provide fundamental insight into the siRNA-mediated RNAi mechanism using a thymoma-derived cell line model to demonstrate for the first time the occurrence of RNAi in primary T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E10 is an immature double-positive thymocyte line derived
from a TCR- and p53 double-mutant mouse of a mixed 129/Sv x
C57BL/6 background as described (23). These cells, which
proliferated vigorously, were maintained at a maximal concentration of
2 x 106 cells/ml and were propagated
in complete medium: DMEM supplemented with 10% heat-inactivated FCS, 2
mM L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 50 µM 2-ME. Cell culture of primary lymphocytes:
cells from the spleen and lymph nodes of DO11.10 TCR-transgenic mice (a
generous gift from Dr. C. London, University of California, Davis, CA)
were activated for 3 days with 1 µg/ml OVA peptide (residues
323–339) in RPMI medium containing 10% FBS.
Transfection
For electroporations, 2.5 µmol dsRNA and/or 20 µg of pEGFP-N3 plasmid (Clontech Laboratories, Palo Alto, CA) were added to prechilled 0.4-cm electrode gap cuvettes (Bio-Rad, Hercules, CA). E10 cells (1.5 x 107) were resuspended to 3 x 107 cells/ml in cold serum-free RPMI, added to the cuvettes, mixed, and pulsed once at 300 mV, 975 µF with a Gene Pulser electroporator II (Bio-Rad). Cells were plated into 6-well culture plates containing 8 ml of complete medium and were incubated at 37°C in a humidified 5% CO2 chamber. Cell viability immediately after electroporation was typically around 60%. For cationic lipid transfections, 2 µg of plasmid DNA and 100 nmol siRNAs were used per 106 cells, and transfection followed manufacturer’s recommended protocol. Transfection of primary lymphocytes: activated DO11.10 T cells were electroporated as above, except that the cells were resuspended to 6 x 107 cells/ml in cold serum-free RPMI and the pulse voltage was 310 mV. After electroporation, the cells were put into four wells of a 24-well plate, each containing 1 ml of RPMI supplemented with 1 ng/ml IL-2 (BioSource International, Camarillo, CA). siRNA oligos (Dharmacon, Lafayette, CO) used were as follows (sense strand is given): effective CD4 siRNA, CD4 no. 4, (sense) gagccauaaucucaucugadgdg, (anti-sense) ucagaugagauuauggcucdtdt; effective CD8 siRNA, CD8 no. 4, (sense) gcuacaacuacuacaugacdtdt, (antisense) gucauguaguaguuguagcdtdt; ineffective siRNAs, CD8 no. 1, (sense) gaaaauggacgccgaacuudgdg, (anti-sense), aaguucggcguccauuuucdtdt; CD8 no. 2, (sense) cgugggacgagaagcugaadtdt, (antisense) uucagcuucucgucccacgdtdt; CD8 no. 3 (sense) aauuguguaaaauggcaccgcdcda, (antisense) µggcggugccauuuuacacaadtdt; CD4 no. 1, (sense) ggagaccaccaugugccgadgdc, (anti-sense) ucggcacaugguggucuccdtdt; CD4 no. 2, (sense) ggcagagaaggauucuuucdtdt, (anti-sense) gaaagaauccuucucugccdtdt; CD4 no. 3, (sense) ccaccugcguccugucucadtdc, (antisense) gugguggacgcaggacagadgdt; CD4 no. 5 (sense) ccaccugcguccugucucadtdc, (antisense) ucagaugagauuauggcucdtdt.
Flow cytometry
E10 cells (
1 x 106) were washed
once in FACS buffer (PBS supplemented with 2% FCS and 0.01% sodium
azide), resuspended to 100 µl, and stained directly with
PE-conjugated anti-CD4 (clone RM4-5) or
allophycocyanin-conjugated anti-CD8 mAbs, and in some
experiments with PE- or allophycocyanin-conjugated anti-mouse
Thy-1.2 (clone 53-2.1) mAb. All mAbs were from BD PharMingen (San
Diego, CA). The stained cells were washed once, then resuspended in 200
µl FACS buffer containing 200 ng/ml propidium iodide (PI). Unstained
and singly stained controls were included in every experiment. 3A9, a T
cell hybridoma line that had been infected with a MIGW green
fluorescent protein (GFP) retrovirus was included when GFP
expression was analyzed. Cell data were collected on a FACSCalibur flow
cytometer (BD Biosciences, San Jose, CA) and four-color analyses (GFP,
PE, PI, and allophycocyanin) were done with CellQuest software (BD
Biosciences). All data were collected by analyses performed on 1
x 104 PI-negative events (viable cells). For the
primary T cell studies, activated cells were analyzed as above, except
that allophycocyanin-conjugated anti-CD4 and PE-conjugated
anti-CD8 were used, and 5 x 104
PI-negative events were analyzed.
Northern blot analysis of mRNA
Cells were lysed in TRIzol reagent (Life Technologies, Grand
Island, NY) and total cellular RNA was purified according to
manufacturer’s instructions. RNA (10 µg) was fractionated on a
denaturing 1% formaldehyde/agarose gel and transferred to a
nitrocellulose membrane. Blots were hybridized overnight with
32P-labeled CD4 (818 bp) or CD8 (596 bp) cDNA
fragments. After washes, blots were analyzed by a PhosphorImager
(Molecular Dynamics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study RNAi, siRNAs are typically delivered into cells by
carrier-mediated transfection reagents. We developed an experimental
system using a thymoma-derived cell line, E10 (23),
wherein we studied the use of siRNAs to silence either CD4 or CD8,
using the other marker as an internal specificity control. However,
typical of lymphocytes, E10 is insensitive to several different
cationic and noncationic transfection reagents and thus electroporation
was used to introduce siRNAs. Using this method, 20% of the cell
population expressed GFP from a transfected reporter vector. When CD4
or CD8 siRNAs were electroporated into E10, a marked reduction in
surface CD4 or CD8 expression, respectively, occurred 36 h
later. Flow cytometry analysis showed that most of the cells were
transfected and expression levels were reduced >5-fold below wild-type
expression levels (Fig. 1
A).
The degree of reduction of CD8 was frequently more pronounced than
that of CD4 and, in both cases, a small population of cells appeared to
be either untransfected or not responsive to the siRNA treatment. In
repeated experiments, typically 70–95% of the cells exhibited a
>5-fold reduction in CD8 expression, although sometimes a smaller
fraction of cells down-regulated CD8 to a greater degree (Fig. 1A).
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2 wk in HeLa cells, although neither the
extent of silencing nor the number of cell divisions was reported. A
time course assay was performed in CD8 siRNA-transfected E10 cells.
GFP was included in these transfections to investigate the relationship
between the uptake and expression of plasmid DNA and siRNAs. Because
these experiments were transient transfections, cell doubling results
in a decrease in GFP fluorescence intensity and number of GFP-positive
cells (Fig. 1
B, NS RNA). When CD8 siRNAs were
cotransfected with the GFP reporter vector, CD8 expression, but not
GFP expression, was markedly reduced (see Fig. 1B, 24
h). Several cell populations were evident, with the major CD8
silenced population displaying >5-fold reduced CD8 expression. The
majority of cells within this population did not express GFP. However,
cells that did express GFP also silenced CD8. This corresponded to
20% of the total cells, similar to the control GFP alone (Fig. 1B, NS siRNA, 24 h). This indicates that all of the
cells expressing GFP also received an adequate level of siRNAs to
silence CD8. In addition, a large fraction of cells incorporated
biologically active levels of siRNAs and yet did not express plasmid
DNA. In this experiment, time points were taken over a period of 6
days. At each time point, one-half of the cells were removed from the
dish and replaced with fresh medium. The collected cells were stained
for CD8 and analyzed by flow cytometry (Fig. 1). A decrease in
CD8 surface expression was detectable at 12 h posttransfection,
with maximal silencing at 36 h. By 96 h, nearly all of the
cells expressed wild-type levels of CD8. Thus, the RNAi effect in
these T cells is a transient phenomena.
In these experiments, there was a dramatic decrease in GFP expression
over time, which was likely a result of dilution of the plasmid or
potentially due to toxicity of high GFP expression. Because 100% of
the GFP-expressing cells exhibited CD8 silencing, it was possible to
monitor the "fate" of this subset of silenced cells. The T cells
that actively underwent CD8 silencing continued to express GFP over
the time course, to the same level as the control population of cells
that were not transfected with siRNAs (compare nonspecific RNA to CD8
siRNA). At 96 h, <5% of the total cells were GFP-positive in
cells treated with nonspecific siRNAs and in CD8 siRNA-treated
samples. These few remaining GFP-positive cells exhibited normal levels
of CD8 expression. This suggests that the cells did not specifically
undergo apoptosis as a result of siRNA transfection and subsequent
CD8 silencing.
Specificity of siRNA-mediated silencing
Although the GFP transgene expression was not affected during
CD8 silencing, the expression of endogenous genes might have been
nonspecifically affected. To address this question, the expression
levels of CD4 and Thy1.2 T cell markers were examined in cells actively
undergoing CD8 silencing. Examination of these markers revealed that
there was no reduction of nontargeted gene expression when compared
with the control nontransfected cells (Fig. 2A), even over extended times
(not shown). Although unlikely for this cell line, an additional
analysis confirmed that the T cells did not become activated, as they
do not up-regulate CD69 (Fig. 2A). Together, these
experiments confirm the specificity of siRNA-mediated CD8
silencing.
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mRNA
Short temporal RNAs such as lin-4 and let-7
mediate silencing by binding to the 3'-untranslated region
(UTR), thus suppressing translation (24, 25, 26). This
is in marked contrast to the posttranscriptional mRNA degradation
effected by siRNAs. To distinguish between these two potential
mechanisms for CD8 silencing, a time course Northern blot analysis
of CD8 mRNA was performed. The process of silencing did not
appreciably affect the growth rate, as compared with control
nonspecific siRNA transfections performed in parallel (not shown). Flow
cytometry analysis indicated that the RNAi response in these cells
lasted 3–4 days (8–10 cell doublings), which corresponds to an
100-fold increase in cell mass (Fig. 2B). Time course
analysis was performed in four independent experiments and expression
of CD8 was typically suppressed 5-fold or greater.
At various time points, a fraction of the cells was used to isolate
total RNA for Northern blot analysis (Fig. 2C). The CD8
mRNA was resolved into two bands, due to alternative splicing
(27, 28). Levels of CD8 mRNA decreased during the
course of CD8 silencing. Densitometric analysis of the CD8 mRNA
bands was performed and normalized to the internal control CD4 band. At
the point of maximal silencing, mRNA levels decrease only 2.5-fold.
This value is not commensurate with the 5-fold decrease in protein
expression determined by the flow cytometric analysis. However, this
RNA was prepared from total cells in which 30% of the cells did not
exhibit any silencing. When corrected for this reduction, CD8 mRNA
was nearly proportionate to levels in reduction of CD8 protein.
These Northern blots were performed multiple times with similar
results. Thus, although it is clear that CD8 mRNA decreases, we
cannot rule out additional silencing phenomena such as cotranslational
repression.
Regional sensitivity of an mRNA to silencing by a siRNA
A major outstanding question is whether any region of a mRNA can
serve as an effective target for siRNA-directed silencing. Several
different siRNAs that targeted different regions of the CD8 mRNA
were tested. Of the first two CD8 siRNAs that were transfected, only
one was active. To more quantitatively examine this difference, cells
were transfected with varying amounts of siRNAs and CD8 expression
was measured by flow cytometry. Cells undergoing silencing were
quantified and compared with control nonspecific siRNA treatment (Fig. 3A). For the effective CD8
siRNA, picomolar amounts were sufficient to induce some silencing and
higher amounts produced a graded response. For the noneffective CD8
siRNA, even at the highest concentration tested, there was no
activity.
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siRNAs were noneffective at silencing. For
CD8, four different siRNAs were synthesized and tested in the flow
cytometry assay: one overlapped the start codon, one which targeted the
open reading frame (ORF), one which overlapped the stop codon,
and one which targeted the 3'-UTR 15 nt after the stop codon. Only the
siRNA which targeted the 3'-UTR 15 nt after the stop codon
effectively silenced CD8 expression. For CD4, five siRNAs were
synthesized which targeted corresponding regions to those for the
CD8 mRNA (Fig. 3B). In this case, only the siRNA that
targeted the stop codon was effective at reducing CD4 expression
levels. An examination of the nucleotide sequences did not reveal any
obvious differences between the effective and ineffective siRNAs.
For each of the above siRNAs, the silencing assay was performed at
different siRNA concentrations. None of the inactive siRNAs generated
detectable silencing at five times the highest concentration of the
active siRNAs (Fig. 3A and data not shown). However, these
inactive siRNAs were able to compete with the silencing of the active
siRNAs. In these competition experiments, inactive CD8 siRNAs were
added into the cuvettes containing the active CD8 siRNA, so that
both could be electroporated into the cells simultaneously. Varying
concentrations were tested, and cells were monitored for CD8
silencing at 36 h (Fig. 4). It was
found that when the total siRNA pool contained an inactive CD4 or
CD8 siRNA, then silencing mediated by an active siRNA was markedly
reduced (Fig. 4, A and B). These results mirror
the ability for active siRNAs to compete for other active siRNAs, a
response that we observed for attempting silencing of both CD4 and
CD8 simultaneously (Fig. 4, C and D). The
inability to silence both CD4 and CD8 simultaneously in the same
cell might suggest that siRNA-mediated RNAi is titratable, as has been
described for silencing using long dsRNAs in C. elegans
(29).
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genes were expressed from CMV-driven
promoters in HeLa cells. The CD8 expression construct contained two
regions that corresponded to target sites for effective and ineffective
siRNAs in E10. In this assay, cationic lipid cotransfection of the
mouse CD4 and CD8 plasmid vectors was performed with either the
effective or noneffective CD8 siRNA. When compared with the
nonspecific siRNA control, CD8-specific RNAi silencing was
recapitulated in HeLa cells, and the ORF-targeted siRNA was still
ineffective at silencing (Fig. 5A). These results suggested
that the noneffective siRNA phenomenon is not unique to the T cell
line, but is likely a feature of either the siRNA sequence, or more
likely the mRNA. The concentration dependence of the effective and
ineffective siRNA was evaluated in the HeLa cell assay. In this
experiment, cationic lipid:siRNA complexes were preformed and added to
the cells as previously described (13). The effective
siRNA exhibited a concentration dependence; however, the ineffective
siRNAs remained inactive even at the highest concentrations (Fig. 5B).
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To test whether primary cells are sensitive to siRNA-mediated
silencing, the CD4/CD8 siRNAs characterized above were used to
silence in primary mouse T cells taken from spleen. In these studies
DO11.10 mice, which express a transgenic TCR that recognizes OVA
peptide in the context of MHC class II were isolated from these mice
are predominantly CD4+; however, a small number
(15%) of CD8+ cells exist in these mice.
Efforts to transfect and silence naive T cells were unsuccessful, but
if the cells were stimulated to divide by the cognate OVA peptide, CD4
and CD8 silencing could be accomplished similar to the E10 thymoma
cell line. Electroporation of CD4 siRNAs into activated primary T cells
resulted in an approximate 5-fold decrease in CD4 surface expression
compared with an unrelated siRNA control (Fig. 6). Costaining for CD8 on the same
cells demonstrated that the down-regulation of CD4 was specific. The
maximal degree of silencing was reached at 48 h posttransfection.
Later time points could not be collected because of reduced cell
viability after 72 h in culture. Similarly, the subset of
CD8-positive T cells electroporated with CD8 siRNA exhibited a maximal
3.3-fold decrease in CD8 levels. Furthermore, the degree of
silencing in the sample population with the alternate coreceptor (i.e.,
CD4 in a CD8 siRNA-treated sample) verified that the RNAi response
was specific (data not shown). These results demonstrate that primary,
mature T cells are able to perform RNAi. The overall degree, kinetics,
and specificity of silencing of CD4 or CD8 in primary T cells was
comparable to that of the E10 cell line, further supporting the
validity of using this line to characterize T cell RNAi.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell surface glycoproteins are of central
importance to immune function and disease. We have quantitatively
tested the efficacy of a variety of siRNAs to suppress the expression
of these glycoproteins. Targeting the CD4 and CD8 markers was
attractive since turnover of coreceptor message is fairly rapid (12
h for CD8), and changes in surface expression can be rapidly and
easily assayed by flow cytometry. In this analysis of two different
genes, we observed that T cells and thymocytic cell lines are amenable
to siRNA-mediated silencing. These studies revealed that siRNA-mediated
RNAi is transient, lasting approximately eight cell doublings. Not
every siRNA was able to induce silencing, and the RNAs which targeted
the 3'-UTR were effective for both genes. Although small temporal RNAs
(stRNAs) mediated translational repression at the mRNA 3'-UTR (for
recent reviews, see Refs. 30, 31, 32, 33, 34), Northern blot analysis
of CD4 and CD8 mRNA indicated posttranscriptional degradation of the
mRNA, consistent with a RNAi-type mechanism of silencing. Finally, in
primary T cells, the overall penetrance and kinetics of CD4 and CD8
siRNA-mediated RNAi was found to be similar to that observed in the E10
thymoma cell line.
In several experiments, and using electroporation, we found efficient
uptake and silencing of >90% of the cells. However, this required the
addition of a relatively high amount of siRNA (2.5 µmol/1.5 x
107 cells); Northern blot analysis indicates that
only a fraction of the siRNAs (3 x 104
siRNAs/cell) become associated with the cells (data not shown). Only a
fraction of the siRNAs that become associated with cells probably are
functional in silencing gene expression. At lower concentrations of
siRNAs, a similar fraction (70–95%) of cells exhibit a reduction in
CD8 expression, albeit at reduced efficiency. Using either
electroporation for T cells or Lipofectamine 2000 for HeLa cells, we
found that 100% of the cells that take up and express a cotransfected
GFP marker also perform RNAi. Based on this fact, it should be possible
to design gene function experiments which enrich the pool of silenced
cells by selecting for the activity of a transfected plasmid
reporter.
Time course analysis of CD8 silencing in the E10 cell line indicated
that the silencing was transient in nature, lasting 3–4 days. As
this cell line doubles rapidly, this value corresponds to approximately
eight cell doublings. Northern blots indicated that silencing
corresponded to a reduction in mRNA levels, commensurate with the
predicted model for RNAi. A translational repression mechanism has been
suggested for silencing mediated by stRNAs via the 3' untranslated
region of developmentally important genes. Although the reduction in
mRNA level approximated that of CD8 expression, we cannot rule out
the possibility of additional translational repression mechanisms.
Only a limited number of the siRNA sequences tested could induce RNAi.
For the silencing of most genes, on average one of two candidate siRNAs
designed is active in contrast to the one of four and one in five
siRNAs tested in targeting CD4 and CD8 (6). It is
interesting to note that the siRNAs that were active in silencing
targeted the 3'-UTR and stop codon. The restrictive utilization of the
3'-UTR siRNAs did not appear to be cell-type specific, as active and
inactive siRNAs gave similar results in HeLa cells. It is unclear why
targeting the mouse CD4 and CD8 mRNA 3'-UTRs were effective for
performing siRNA-mediated RNAi, while other sites were not. One
possibility is that further testing of other mRNA regions would result
in productive silencing (35). Alternatively, perhaps the
3'-UTR of these genes is particularly accessible for targeting.
Silencing of developmentally timed genes in the endogenous stRNA
pathway is specific for the 3'-UTR (25, 36). This could be
a common feature of developmentally timed genes, because both CD4 and
CD8 are also expressed in a developmentally timed manner.
Attempting to silence both CD4 and CD8 simultaneously resulted in
lower levels of silencing of each gene. These results supports a
previously recognized observation that the RNAi response is titratable
(29). Surprisingly, several of the siRNAs that were
inactive competed for silencing when coelectroporated with active
siRNAs. While this manuscript was in preparation, another group
reported similar findings for the silencing of human coagulation
trigger factor (37). However, another group has reported
success in dual gene targeting of Lamin A/C and NuMA proteins in HeLa
cells (38). The data presented in this study indicate that
the inactive siRNAs are recognized by cellular processes but either
cannot be converted to an active structure for gene silencing or cannot
gain access to their complementary sequences on the target
mRNA.
This work presents the first evidence for silencing by siRNA in primary
somatic mammalian lymphocytes. In these studies, the degree and
kinetics of CD4 and CD8 silencing in the activated primary cells was
similar to that of the E10 cell line. In both the primary cells and E10
cells the onset of maximal silencing appeared around three to four cell
doublings, which corresponded to 36–48 h posttransfection. In the E10
cells, 100% of the cells had resumed normal CD8 expression by
96 h. Because the viability of the primary cells began to diminish
at around 60 h, it was difficult to determine how long the RNAi
response would last past 72 h. It is interesting to note that the
cells needed to be activated in order for silencing to be accomplished.
This could be due to the inability to take up the siRNAs after
electroporation, as primary T cells are known to be difficult to
transfect with nucleic acids. It is unknown whether mammalian cells
must be in a dividing, or "competent", state to perform RNAi.
Future studies of siRNA-mediated RNAi in primary cells are required to
distinguish between these two possibilities. Nevertheless, these
findings provide a precedent upon which future studies of T lymphocyte
biology can be designed to validate function by siRNA-mediated
silencing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Phillip A. Sharp, Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames Street E17-526, Cambridge, MA 02139. E-mail address: sharppa{at}mit.edu
3 Abbreviations used in this paper: RNAi, RNA interference; siRNA, small interfering RNA; PI, propidium iodide; GFP, green fluorescent protein; ORF, open reading frame; UTR, untranslated region; stRNA, small temporal RNA.
Received for publication May 28, 2002. Accepted for publication September 12, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,3-galactosyltransferase family 3GalT6). J. Biol. Chem. 276:48189.: characterization of the human CD8 gene. J. Immunol. 142:3312.[Abstract]
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R. Piva, R. Chiarle, A. D. Manazza, R. Taulli, W. Simmons, C. Ambrogio, V. D'Escamard, E. Pellegrino, C. Ponzetto, G. Palestro, et al. Ablation of oncogenic ALK is a viable therapeutic approach for anaplastic large-cell lymphomas Blood, January 15, 2006; 107(2): 689 - 697. [Abstract] [Full Text] [PDF]
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S. K. Pathak, S. Basu, A. Bhattacharyya, S. Pathak, M. Kundu, and J. Basu Mycobacterium tuberculosis Lipoarabinomannan-mediated IRAK-M Induction Negatively Regulates Toll-like Receptor-dependent Interleukin-12 p40 Production in Macrophages J. Biol. Chem., December 30, 2005; 280(52): 42794 - 42800. [Abstract] [Full Text] [PDF]
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T. Methi, J. Ngai, M. Mahic, M. Amarzguioui, T. Vang, and K. Tasken Short-Interfering RNA-Mediated Lck Knockdown Results in Augmented Downstream T Cell Responses J. Immunol., December 1, 2005; 175(11): 7398 - 7406. [Abstract] [Full Text] [PDF]
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C. A. Sledz and B. R. G. Williams RNA interference in biology and disease Blood, August 1, 2005; 106(3): 787 - 794. [Abstract] [Full Text] [PDF]
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P. Oberdoerffer, C. Kanellopoulou, V. Heissmeyer, C. Paeper, C. Borowski, I. Aifantis, A. Rao, and K. Rajewsky Efficiency of RNA Interference in the Mouse Hematopoietic System Varies between Cell Types and Developmental Stages Mol. Cell. Biol., May 15, 2005; 25(10): 3896 - 3905. [Abstract] [Full Text] [PDF]
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J. L. Round, T. Tomassian, M. Zhang, V. Patel, S. P. Schoenberger, and M. C. Miceli Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells J. Exp. Med., February 7, 2005; 201(3): 419 - 430. [Abstract] [Full Text] [PDF]
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E. Agbottah, C. de La Fuente, S. Nekhai, A. Barnett, A. Gianella-Borradori, A. Pumfery, and F. Kashanchi Antiviral Activity of CYC202 in HIV-1-infected Cells J. Biol. Chem., January 28, 2005; 280(4): 3029 - 3042. [Abstract] [Full Text] [PDF]
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M. K. Saifullah, D. A. Fox, S. Sarkar, S. M. A. Abidi, J. Endres, J. Piktel, T. M. Haqqi, and N. G. Singer Expression and Characterization of a Novel CD6 Ligand in Cells Derived from Joint and Epithelial Tissues J. Immunol., November 15, 2004; 173(10): 6125 - 6133. [Abstract] [Full Text] [PDF]
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F.-Q. Wen, X. Liu, T. Kobayashi, S. Abe, Q. Fang, T. Kohyama, R. Ertl, Y. Terasaki, L. Manouilova, and S. I. Rennard Interferon-{gamma} Inhibits Transforming Growth Factor-{beta} Production in Human Airway Epithelial Cells by Targeting Smads Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 816 - 822. [Abstract] [Full Text] [PDF]
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Y.-L. Chiu, H. Cao, J.-M. Jacque, M. Stevenson, and T. M. Rana Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference Directed against Human Transcription Elongation Factor P-TEFb (CDK9/CyclinT1) J. Virol., March 1, 2004; 78(5): 2517 - 2529. [Abstract] [Full Text] [PDF]
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 J. A. Davies, M. Ladomery, P. Hohenstein, L. Michael, A. Shafe, L. Spraggon, and N. Hastie Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation Hum. Mol. Genet., January 15, 2004; 13(2): 235 - 246. [Abstract] [Full Text] [PDF]
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M. Ehlers, K. Laule-Kilian, M. Petter, C. J. Aldrian, B. Grueter, A. Wurch, N. Yoshida, T. Watanabe, M. Satake, and V. Steimle Morpholino Antisense Oligonucleotide-Mediated Gene Knockdown During Thymocyte Development Reveals Role for Runx3 Transcription Factor in CD4 Silencing During Development of CD4-/CD8+ Thymocytes J. Immunol., October 1, 2003; 171(7): 3594 - 3604. [Abstract] [Full Text] [PDF]
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D. Laderach, D. Compagno, O. Danos, W. Vainchenker, and A. Galy RNA Interference Shows Critical Requirement for NF-{kappa}B p50 in the Production of IL-12 by Human Dendritic Cells J. Immunol., August 15, 2003; 171(4): 1750 - 1757. [Abstract] [Full Text] [PDF]
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N. Bidere, H. K. Lorenzo, S. Carmona, M. Laforge, F. Harper, C. Dumont, and A. Senik Cathepsin D Triggers Bax Activation, Resulting in Selective Apoptosis-inducing Factor (AIF) Relocation in T Lymphocytes Entering the Early Commitment Phase to Apoptosis J. Biol. Chem., August 15, 2003; 278(33): 31401 - 31411. [Abstract] [Full Text] [PDF]
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S. Matsukura, P. A. Jones, and D. Takai Establishment of conditional vectors for hairpin siRNA knockdowns Nucleic Acids Res., August 1, 2003; 31(15): e77 - e77. [Abstract] [Full Text] [PDF]
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J. A. Hill, T. E. Ichim, K. P. Kusznieruk, M. Li, X. Huang, X. Yan, R. Zhong, E. Cairns, D. A. Bell, and W.-P. Min Immune Modulation by Silencing IL-12 Production in Dendritic Cells Using Small Interfering RNA J. Immunol., July 15, 2003; 171(2): 691 - 696. [Abstract] [Full Text] [PDF]
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D. Semizarov, L. Frost, A. Sarthy, P. Kroeger, D. N. Halbert, and S. W. Fesik Specificity of short interfering RNA determined through gene expression signatures PNAS, May 27, 2003; 100(11): 6347 - 6352. [Abstract] [Full Text] [PDF]
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T. Holen, M. Amarzguioui, E. Babaie, and H. Prydz Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway Nucleic Acids Res., May 1, 2003; 31(9): 2401 - 2407. [Abstract] [Full Text] [PDF]
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); two ineffective siRNAs (
, 
).
B, Relative target locations of the siRNAs used in these
studies. Schematic of CD4 and CD8
