letters to nature
`
`ogy), respectively. Staining specificity was controlled by single staining, as well as by using
`secondary antibodies in the absence of the primary stain.
`
`Generation of target cells
`Target cells displaying a membrane-integral version of either wild-type HEL or a mutant10
`exhibiting reduced affinity for HyHEL10 ([R21, D101, G102, N103] designated HEL*) were
`generated by transfecting mouse J558L plasmacytoma cells with constructs analogous to
`those used10 for expression of soluble HEL/HEL*, except that 14 Ser/Gly codons, the H2Kb
`transmembrane region, and a 23-codon cytoplasmic domain were inserted immediately
`upstream of the termination codon by polymerase chain reaction. For mHEL–GFP, we
`included the EGFP coding domain in the Ser/Gly linker. Abundance of surface HEL was
`monitored by flow cytometry and radiolabelled-antibody binding using HyHEL5 and
`D1.3 HEL-specific monoclonal antibodies, for which the mutant HELs used in this work
`show unaltered affinities10.
`
`Interaction assays
`For B-cell/target interaction assays, splenic B cells from 3-83 or MD4 transgenic mice28,29
`carrying (IgM + IgD) BCRs specific for HEL or H2Kk/H2Kbwere freshly purified on
`Lympholyte and incubated with a twofold excess of target cells in RPMI, 50 mM HEPES
`pH 7.4, for the appropriate time at 37 8C before being applied to polylysine-coated slides.
`Cells were fixed in 4% paraformaldehyde/PBS or methanol and permeabilized with PBS/
`0.1% Triton X-100 before immunofluorescence. We acquired confocal images using a
`Nikon E800 microscope attached to BioRad Radiance Plus scanning system equipped with
`488-nm and 543-nm lasers, as well as differential interference contrast for transmitted
`light. GFP fluorescence in living cells in real time was visualized using a Radiance 2000 and
`Nikon E300 inverted microscope. Images were processed using BioRad Lasersharp 1024 or
`2000 software to provide single plane images, confocal projections or slicing.
`
`Antigen presentation
`Presentation of HEL epitopes to T-cell hybridomas 2G7 (specific for I-Ek[HEL1–18]) and
`1E5 (specific for I-Ed[HEL108–116]) by transfectants of the LK35.2 B-cell hybridoma
`expressing an HEL-specific IgM BCR was monitored as described10.
`
`Received 12 December 2000; accepted 30 March 2001.
`
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`5. Schamel, W. W. & Reth, M. Monomeric and oligomeric complexes of the B cell antigen receptor.
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`J. Exp. Med. 191, 1443–1448 (2000).
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`14. Lang, J. et al. B cells are exquisitely sensitive to central tolerance and receptor editing by ultralow
`affinity, membrane-bound antigen. J. Exp. Med. 184, 1685–1697 (1996).
`15. Valitutti, S., Muller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of many T-cell
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`Science 285, 221–227 (1999).
`19. Leupin, O., Zaru, R., Laroche, T., Muller, S. & Valitutti, S. Exclusion of CD45 from the T-cell receptor
`signaling area in antigen-stimulated T lymphocytes. Curr. Biol. 10, 277–280 (2000).
`20. Cagan, R. L., Kramer, H., Hart, A. C. & Zipursky, S. L. The bride of sevenless and sevenless interaction:
`internalization of a transmembrane ligand. Cell 69, 393–399 (1992).
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`surface molecules derived from antigen-presenting cells. J. Exp. Med. 191, 1137–1148 (2000).
`23. Batista, F. D. & Neuberger, M. S. B cells extract and present immobilized antigen: implications for
`affinity discrimination. EMBO J. 19, 513–520 (2000).
`24. Casten, L. A., Lakey, E. K., Jelachich, M. L., Margoliash, E. & Pierce, S. K. Anti-immunoglobulin
`
`augments the B-cell antigen-presentation function independently of internalization of receptor-
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`antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160, 5203–5208
`(1998).
`26. Serre, K. et al. Efficient presentation of multivalent antigens targeted to various cell surface molecules
`of dendritic cells and surface Ig of antigen-specific B cells. J. Immunol. 161, 6059–6067 (1998).
`27. Green, S. M., Lowe, A. D., Parrington, J. & Karn, J. Transformation of growth factor-dependent
`myeloid stem cells with retroviral vectors carrying c-myc. Oncogene 737–751 (1989).
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`29. Goodnow, C. C. et al. Altered immunoglobulin expression and functional silencing of self-reactive B
`lymphocytes in transgenic mice. Nature 334, 676–682 (1988).
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`Supplementary information is available on Nature’s World-Wide Web site
`(http://www.nature.com) or as paper copy from the London editorial office of Nature.
`
`Acknowledgements
`We thank B. Amos and S. Reichelt for help and advice with confocal microscopy, and
`S. Munro for helpful discussions. We are indebted those who provided antibodies,
`transgenic mice and cell lines. F.D.B. and D.I. were supported by the Arthritis Research
`Campaign and Studienstiftung des deutschen Volkes, respectively.
`
`Correspondence and requests for materials should be addressed to F.D.B.
`(e-mail:fdb@mrc-lmb.cam.ac.uk) or M.S.N. (e-mail:msn@mrc-lmb.cam.ac.uk)
`
`.................................................................
`Duplexes of 21–nucleotide RNAs
`mediate RNA interference in
`cultured mammalian cells
`
`Sayda M. Elbashir*, Jens Harborth†, Winfried Lendeckel*,
`Abdullah Yalcin*, Klaus Weber† & Thomas Tuschl*
`
`* Department of Cellular Biochemistry; and † Department of Biochemistry and
`Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11,
`D-37077 Go¨ttingen, Germany
`
`..............................................................................................................................................
`RNA interference (RNAi) is the process of sequence-specific,
`post-transcriptional gene silencing in animals and plants,
`initiated by double-stranded RNA (dsRNA) that is homologous
`in sequence to the silenced gene1–4. The mediators of sequence-
`specific messenger RNA degradation are 21- and 22-nucleotide
`small interfering RNAs (siRNAs) generated by ribonuclease III
`cleavage from longer dsRNAs5–9. Here we show that 21-nucleo-
`tide siRNA duplexes specifically suppress expression of endo-
`genous and heterologous genes in different mammalian cell
`lines,
`including human embryonic kidney (293) and HeLa
`cells. Therefore, 21-nucleotide siRNA duplexes provide a new
`tool for studying gene function in mammalian cells and may
`eventually be used as gene-specific therapeutics.
`Uptake of dsRNA by insect cell lines has previously been shown to
`‘knock-down’
`the expression of
`specific proteins, owing to
`sequence-specific, dsRNA-mediated mRNA degradation6,10–12.
`However, it has not been possible to detect potent and specific
`RNA interference in commonly used mammalian cell culture
`systems,
`including 293 (human embryonic kidney), NIH/3T3
`(mouse fibroblast), BHK-21 (Syrian baby hamster kidney), and
`CHO-K1 (Chinese hamster ovary) cells, applying dsRNA that varies
`in size between 38 and 1,662 base pairs (bp)10,12. This apparent lack
`of RNAi in mammalian cell culture was unexpected, because RNAi
`exists in mouse oocytes and early embryos13,14, and because RNAi-
`related, transgene-mediated co-suppression was also observed in
`cultured Rat-1 fibroblasts15. But it is known that dsRNA in the
`cytoplasm of mammalian cells can trigger profound physiological
`
`494
`
`© 2001 Macmillan Magazines Ltd
`
`NATURE | VOL 411 | 24 MAY 2001 | www.nature.com
`
`Benitec - Exhibit 1016 - page 1
`
`

`
`letters to nature
`
`to 12-fold, GL3 expression 9- to 25-fold, and RL expression 2- to 3-
`fold, in response to the cognate siRNAs. For 293 cells, targeting of
`RL luciferase by RL siRNAs was ineffective, although GL2 and GL3
`targets responded specifically (Fig. 2i and j). The lack of reduction of
`RL expression in 293 cells may be because of its expression, 5- to 20-
`fold higher than any other mammalian cell line tested and/or to
`limited accessibility of the target sequence due to RNA secondary
`structure or associated proteins. Nevertheless, specific targeting of
`GL2 and GL3 luciferase by the cognate siRNA duplexes indicated
`that RNAi is also functioning in 293 cells.
`The 2-nucleotide 39 overhang in all siRNA duplexes was com-
`posed of (29-deoxy) thymidine, except for uGL2, which contained
`
`S2, pGL3-Control/pRL-TK
`
`Norm. Rr-luc/Pp-luc
`
`NIH/3T3
`
`COS-7
`
`HeLa S3
`
`293
`
`Bu uGL2 GL2 GL3 invGL2 RL
`siRNA
`
`b
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`d
`1.6
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`f
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`h
`1.6
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`j
`1.2
`1
`0.8
`
`0.6
`0.4
`0.2
`0
`
`S2, pGL2-Control/pRL-TK
`
`NIH/3T3
`
`COS-7
`
`HeLa S3
`
`293
`
`Bu uGL2 GL2 GL3 invGL2 RL
`siRNA
`
`a
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`c
`1.6
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`e
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`g
`1.6
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`i
`1.2
`1
`0.8
`
`0.6
`0.4
`0.2
`0
`
`Norm. Pp-luc/Rr-luc
`
`Figure 2 RNA interference by siRNA duplexes. Ratios of target to control luciferase were
`normalized to a buffer control (Bu, black bars); grey bars indicate ratios of Photinus pyralis
`(Pp-luc) GL2 or GL3 luciferase to Renilla reniformis (Rr-luc) RL luciferase (left axis), white
`bars indicate RL to GL2 or GL3 ratios (right axis). a, c, e, g and i, Experiments performed
`with the combination of pGL2-Control and pRL-TK reporter plasmids; b, d, f, h and j,
`experiments performed with the combination of pGL3-Control and pRL-TK reporter
`plasmids. The cell line used for the interference experiment is indicated at the top of each
`plot. The ratios of Pp-luc/Rr-luc for the buffer control (Bu) varied between 0.5 and 10 for
`pGL2/pRL, and between 0.03 and 1 for pGL3/pRL, respectively, before normalization and
`between the various cell lines tested. The plotted data were averaged from three
`independent experiments 6 s.d.
`
`reactions that lead to the induction of interferon synthesis16. In
`the interferon response, dsRNA . 30 bp binds and activates the
`protein kinase PKR17 and 29,59-oligoadenylate synthetase (29,59-
`AS)18. Activated PKR stalls translation by phosphorylation of the
`translation initiation factors eIF2a, and activated 29,59-AS causes
`mRNA degradation by 29,59-oligoadenylate-activated ribonuclease
`L. These responses are intrinsically sequence-nonspecific to the
`inducing dsRNA.
`Base-paired 21- and 22-nucleotide (nt) siRNAs with overhanging
`39 ends mediate efficient sequence-specific mRNA degradation in
`lysates prepared from Drosophila embryos9. To test whether siRNAs
`are also capable of mediating RNAi in cell culture, we synthesized
`21-nt siRNA duplexes with symmetric 2-nt 39 overhangs directed
`against reporter genes coding for sea pansy (Renilla reniformis, RL)
`and two sequence variants of firefly (Photinus pyralis, GL2 and GL3)
`luciferases (Fig. 1a, b). The siRNA duplexes were co-transfected
`with the reporter plasmid combinations pGL2/pRL or pGL3/pRL,
`into Drosophila S2 cells or mammalian cells using cationic lipo-
`somes. Luciferase activities were determined 20 h after transfection.
`In Drosophila S2 cells (Fig. 2a and b), the specific inhibition of
`luciferases was complete and similar to results previously obtained
`for longer dsRNAs6,10,12,19. In mammalian cells, where the reporter
`genes were 50- to 100-fold more strongly expressed, the specific
`suppression was less complete (Fig. 2c–j). In NIH/3T3, monkey
`COS-7 and Hela S3 cells (Fig. 2c–h), GL2 expression was reduced 3-
`
`a
`SV40 prom.
`
`Pp-luc (GL2-SV40)
`
`3'-UTR
`
`enh.
`
`SV40 prom.
`
`poly(A) enh.
`
`Pp-luc (GL3-SV40)
`
`TK prom.
`
`b
`
`siRNA
`duplex
`
`uGL2
`
`GL2
`
`GL3
`
`poly(A)
`Rr-luc (RL-TK)
`
`5'-CGUACGCGGAAUACUUCGAUU
` UUGCAUGCGCCUUAUGAAGCU-5'
`
`5'-CGUACGCGGAAUACUUCGATT
` TTGCAUGCGCCUUAUGAAGCU-5'
`
`5'-CUUACGCUGAGUACUUCGATT
` TTGAAUGCGACUCAUGAAGCU-5'
`
`invGL2
`
`5'-AGCUUCAUAAGGCGCAUGCTT
` TTUCGAAGUAUUCCGCGUACG-5'
`
`RL
`
`5'-AAACAUGCAGAAAAUGCUGTT
` TTUUUGUACGUCUUUUACGAC-5'
`
`Figure 1 Reporter constructs and siRNA duplexes. a, The firefly (Pp-luc) and sea pansy
`(Rr-luc) luciferase reporter-gene regions from plasmids pGL2-Control, pGL3-Control, and
`pRL-TK (Promega) are illustrated; simian virus 40 (SV40) promoter (prom.); SV40
`enhancer element (enh.); SV40 late polyadenylation signal (poly(A)); herpes simplex virus
`(HSV) thymidine kinase promoter, and two introns (lines) are indicated. The sequence of
`GL3 luciferase is 95% identical to GL2, but RL is completely unrelated to both. Luciferase
`expression from pGL2 is approximately 10-fold lower than from pGL3 in transfected
`mammalian cells. The region targeted by the siRNA duplexes is indicated as black bar
`below the coding region of the luciferase genes. b, The sense (top) and antisense (bottom)
`sequences of the siRNA duplexes targeting GL2, GL3, and RL luciferase are shown. The
`GL2 and GL3 siRNA duplexes differ by only three single-nucleotide substitutions (boxed in
`grey). As nonspecific control, a duplex with the inverted GL2 sequence, invGL2, was
`synthesized. The 2-nucleotide 39 overhang of 29-deoxythymidine is indicated as TT; uGL2
`is similar to GL2 siRNA but contains ribo-uridine 39 overhangs.
`
`NATURE | VOL 411 | 24 MAY 2001 | www.nature.com
`
`© 2001 Macmillan Magazines Ltd
`
`495
`
`Benitec - Exhibit 1016 - page 2
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`

`
`shown in Fig. 4, the expression of lamin A/C was specifically
`reduced by the cognate siRNA duplex (Fig. 4a), but not when
`nonspecific siRNA directed against firefly luciferase (Fig. 4b) or
`buffer (Fig. 4c) was used. The expression of a non-targeted gene,
`NuMA, was unaffected in all treated cells (Fig. 4d–f), demonstrat-
`ing the integrity of the targeted cells. The reduction in lamin A/C
`proteins was more than 90% complete as quantified by western
`blotting (Fig. 4j, k). We note that lamin A/C ‘knock-out’ mice are
`
`pGL2-Control/pRL-TK
`
`Bu GL2 GL3 RL
`siRNAs
`
`hG
`50
`
`GL2
`49
`
`GL3
`49
`
`RL
`50
`
`hG
`501
`
`GL2
`484
`
`GL3
`484
`
`RL
`501
`
`Bu GL2 GL3 RL
`siRNAs
`
`hG
`50
`
`GL2
`49
`
`GL3
`49
`
`RL
`50
`
`hG
`501
`
`GL2
`484
`
`GL3
`484
`
`RL
`501
`
`Norm. Rr-luc/Pp-luc
`
`Bu GL2 GL3 RL
`siRNAs
`
`hG
`50
`
`GL2
`49
`
`GL3
`49
`
`RL
`50
`
`hG
`501
`
`GL2
`484
`
`GL3
`484
`
`RL
`501
`
`a
`
`108
`
`107
`
`106
`
`105
`
`104
`
`103
`
`Pp-luc (a.u.)
`
`b
`
`107
`
`106
`
`105
`
`104
`
`103
`
`Rr-luc (a.u.)
`
`c
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`
`Norm. Pp-luc/Rr-luc
`
`Figure 3 Effects of 21-nucleotide siRNAs, 50-bp, and 500-bp dsRNAs on luciferase
`expression in HeLa cells. The exact length of the long dsRNAs in base pairs is indicated
`below the bars. Experiments were performed with pGL2-Control and pRL-TK reporter
`plasmids. The data were averaged from two independent experiments 6 s.d. a, Absolute
`Pp-luc expression, plotted in arbitrary luminescence units (a.u.). b, Rr-luc expression,
`plotted in arbitrary luminescence units. c, Ratios of normalized target to control luciferase.
`The ratios of luciferase activity for siRNA duplexes were normalized to a buffer control (Bu,
`black bars); the luminescence ratios for 50- or 500-bp dsRNAs were normalized to the
`respective ratios observed for 50- and 500-bp dsRNAs from humanized GFP (hG, black
`bars). We note that the overall differences in sequence between the 49- and 484-bp GL2
`and GL3 dsRNAs are not sufficient to confer specificity for targeting GL2 and GL3 targets
`(43-nucleotide uninterrupted identity in 49-bp segment, 239-nucleotide longest
`uninterrupted identity in 484-bp segment)30.
`
`letters to nature
`
`uridine residues. The thymidine overhang was chosen because it
`reduces costs of RNA synthesis and may enhance nuclease resistance
`of siRNAs in the cell culture medium and within transfected cells. As
`in the Drosophila in vitro system (data not shown), substitution of
`uridine by thymidine in the 39 overhang was well tolerated in
`cultured mammalian cells (Fig. 2a, c, e, g and i), and the sequence
`of the overhang appears not to contribute to target recognition9.
`In co-transfection experiments, 25 nM siRNA duplexes were
`used (Figs 2 and 3; concentration is in respect to the final volume
`of tissue culture medium). Increasing the siRNA concentration to
`100 nM did not enhance the specific silencing effects, but started
`to affect transfection efficiencies, perhaps due to competition for
`liposome encapsulation between plasmid DNA and siRNA (data
`not shown). Decreasing the siRNA concentration to 1.5 nM did
`not reduce the specific silencing effect (data not shown), even
`though the siRNAs were now only 2- to 20-fold more concen-
`trated than the DNA plasmids; the silencing effect only vanishes
`completely if
`the siRNA concentration was dropped below
`0.05 nM. This indicates that siRNAs are extraordinarily powerful
`reagents for mediating gene silencing, and that siRNAs are
`effective at concentrations that are several orders of magnitude
`below the concentrations applied in conventional antisense or
`ribozyme gene-targeting experiments20.
`To monitor the effect of longer dsRNAs on mammalian cells, 50-
`and 500-bp dsRNAs that are cognate to the reporter genes were
`prepared. As a control for nonspecific inhibition, dsRNAs from
`humanized GFP (hG)21 was used. In these experiments, the reporter
`plasmids were co-transfected with either 0.21 mg siRNA duplexes or
`0.21 mg longer dsRNAs. The siRNA duplexes only reduced the
`expression of
`their cognate reporter gene, while the longer
`dsRNAs strongly and nonspecifically reduced reporter-gene expres-
`sion. The effects are illustrated for HeLa S3 cells as a representative
`example (Fig. 3a and b). The absolute luciferase activities were
`decreased nonspecifically 10- to 20-fold by 50-bp dsRNA, and 20- to
`200-fold by 500-bp dsRNA co-transfection, respectively. Similar
`nonspecific effects were observed for COS-7 and NIH/3T3 cells. For
`293 cells, a 10- to 20-fold nonspecific reduction was observed only
`for 500-bp dsRNAs. Nonspecific reduction in reporter-gene expres-
`sion by dsRNA . 30 bp was expected as part of the interferon
`response16. Interestingly, superimposed on the nonspecific inter-
`feron response, we detect additional sequence-specific, dsRNA-
`mediated silencing. The sequence-specific silencing effect of long
`dsRNAs, however, became apparent only when the relative reporter-
`gene activities were normalized to the hG dsRNA controls (Fig. 3c).
`Sequence-specific silencing by 50- or 500-bp dsRNAs reduced the
`targeted reporter-gene expression by an additional 2- to 5-fold.
`Similar effects were also detected in the other three mammalian cell
`lines tested (data not shown). Specific silencing effects with dsRNAs
`(356–1,662 bp) were previously reported in CHO-K1 cells, but the
`amounts of dsRNA required to detect a 2- to 4-fold specific
`reduction were about 20-fold higher than in our experiments12.
`Also, CHO-K1 cells appear to be deficient in the interferon
`response. In another report, 293, NIH/3T3 and BHK-21 cells were
`tested for RNAi using luciferase/b-galactosidase (lacZ) reporter
`combinations and 829-bp specific lacZ or 717-bp nonspecific
`green fluorescent protein (GFP) dsRNA10. The lack of detected
`RNAi in this case may be due to the less sensitive luciferase/lacZ
`reporter assay and the length differences of target and control
`dsRNA. Taken together, our results indicate that RNAi is active in
`mammalian cells, but that the silencing effect is difficult to detect if
`the interferon system is activated by dsRNA . 30 bp.
`To test for silencing of endogenous genes, we chose four genes
`coding for cytoskeletal proteins: lamin A/C, lamin B1, nuclear
`mitotic apparatus protein (NuMA) and vimentin27. The selection
`was based on the availability of antibodies needed to quantitate the
`silencing effect. Silencing was monitored 40 to 45 h after transfec-
`tion to allow for turnover of the protein of the targeted genes. As
`
`496
`
`© 2001 Macmillan Magazines Ltd
`
`NATURE | VOL 411 | 24 MAY 2001 | www.nature.com
`
`Benitec - Exhibit 1016 - page 3
`
`

`
`letters to nature
`
`nucleotide siRNAs24. Methylation of promoter regions can lead to
`transcriptional silencing25, but methylation in coding sequences
`does not26. DNA methylation and transcriptional silencing in
`mammals are well documented processes27, yet their mechanisms
`have not been linked to that of post-transcriptional silencing.
`Methylation in mammals is predominantly directed towards CpG
`dinucleotide sequences. There is no CpG sequence in the RL or
`lamin A/C siRNA, although both siRNAs mediate specific silencing
`in mammalian cell culture, so it is unlikely that DNA methylation is
`essential for the silencing process.
`Thus we have shown, for the first time, siRNA-mediated gene
`silencing in mammalian cells. The use of exogenous 21-nucleotide
`siRNAs holds great promise for analysis of gene function in human
`cell culture and the development of gene-specific therapeutics. It
`will also be of
`interest
`in understanding the potential role
`of endogenous siRNAs in the regulation of mammalian gene
`function.
`M
`
`Methods
`RNA preparation
`21-nucleotide RNAs were chemically synthesized using Expedite RNA phosphoramidites
`and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides were
`deprotected and gel-purified9. The accession numbers given below are from GenBank. The
`siRNA sequences targeting GL2 (Acc. No. X65324) and GL3 luciferase (Acc. No. U47296)
`corresponded to the coding regions 153–173 relative to the first nucleotide of the start
`codon; siRNAs targeting RL (Acc. No. AF025846) corresponded to region 119–139 after
`the start codon. The siRNA sequence targeting lamin A/C (Acc. No. X03444) was from
`position 608–630 relative to the start codon; lamin B1 (Acc. No. NM_005573) siRNA was
`from position 672–694; NuMA (Acc. No. Z11583) siRNA from position 3,988–4,010, and
`vimentin (Acc. No. NM_003380) from position 346–368 relative to the start codon.
`Longer RNAs were transcribed with T7 RNA polymerase from polymerase chain reaction
`(PCR) products, followed by gel purification. The 49- and 484-bp GL2 or GL3 dsRNAs
`corresponded to positions 113–161 and 113–596, respectively, relative to the start of
`translation; the 50- and 501-bp RL dsRNAs corresponded to position 118–167 and 118–
`618, respectively. PCR templates for dsRNA synthesis targeting humanized GFP (hG) were
`amplified from pAD3 (ref. 21), whereby 50- and 501-bp hG dsRNA corresponded to
`positions 121–170 and 121–621, respectively, to the start codon.
`For annealing of siRNAs, 20 mM single strands were incubated in annealing buffer
`(100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate)
`for 1 min at 90 8C followed by 1 h at 37 8C. The 37 8C incubation step was extended
`overnight for the 50- and 500-bp dsRNAs, and these annealing reactions were performed
`at 8.4 mM and 0.84 mM strand concentrations, respectively.
`
`Cell culture
`S2 cells were propagated in Schneider’s Drosophila medium (Life Technologies) supple-
`mented with 10% fetal bovine serum (FBS) 100 units ml-1 penicillin, and 100 mg ml-1
`streptomycin at 25 8C. 293, NIH/3T3, HeLa S3, HeLa SS6, COS-7 cells were grown at 37 8C
`in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 units ml-1
`penicillin, and 100 mg ml-1 streptomycin. Cells were regularly passaged to maintain
`exponential growth. Twenty-four h before transfection at 50–80% confluency, mamma-
`lian cells were trypsinized and diluted 1:5 with fresh medium without antibiotics
`(1–3 3 105 cells ml 2 1) and transferred to 24-well plates (500 ml per well). S2 cells were not
`trypsinized before splitting. Co-transfection of reporter plasmids and siRNAs was carried
`out with Lipofectamine 2000 (Life Technologies) as described by the manufacturer for
`adherent cell lines. Per well, 1.0 mg pGL2-Control (Promega) or pGL3-Control
`(Promega), 0.1 mg pRL-TK (Promega), and 0.21 mg siRNA duplex or dsRNA, formulated
`into liposomes, were applied; the final volume was 600 ml per well. Cells were incubated
`20 h after transfection and appeared healthy thereafter. Luciferase expression was
`subsequently monitored with the Dual luciferase assay (Promega). Transfection
`efficiencies were determined by fluorescence microscopy for mammalian cells lines after
`co-transfection of 1.1 mg hGFP-encoding pAD3 (ref. 21) and 0.21 mg inverted GL2 siRNA,
`and were 70–90%. Reporter plasmids were amplified in XL-1 Blue (Stratagene) and
`purified using the Qiagen EndoFree Maxi Plasmid Kit.
`Transfection of siRNAs for targeting endogenous genes was carried out using
`Oligofectamine (Life Technologies) and 0.84 mg siRNA duplex per well, but it was recently
`found that as little as 0.01 mg siRNAs per well are sufficient to mediate silencing. HeLa SS6
`cells were transfected one to three times in approximately 15 h intervals and were assayed
`40 to 45 h after the first transfection. It appears, however, that a single transfection is as
`efficient as multiple transfections. Transfection efficiencies as determined by immuno-
`fluorescence of targeted cells were in the range of 90%. Specific silencing of targeted genes
`was confirmed by at least three independent experiments.
`
`Western blotting and immunofluorescence microscopy
`Monoclonal 636 lamin A/C specific antibody28 was used as undiluted hybridoma super-
`natant for immunofluorescence and 1/100 dilution for western blotting. Affinity-purified
`polyclonal NuMA protein 705 antibody29 was used at a concentration of 10 mg ml-1 for
`
`viable for a few weeks after birth23 and that the lamin A/C knock-
`down in cultured cells was not expected to cause cell death. Lamin A
`and C are produced by alternative splicing in the 39 region and
`are present in equal amounts in the lamina of mammalian cells
`(Fig. 4j, k). Transfection of siRNA duplexes targeting lamin B1 and
`NuMA reduced the expression of these proteins to low levels (data
`not shown), but we were not able to observe a reduction in vimentin
`expression. This could be due to the high abundance of vimentin in
`the cells (several per cent of total cell mass) or because the siRNA
`sequence chosen was not optimal for targeting of vimentin.
`The mechanism of the 21-nucleotide siRNA-mediated interfer-
`ence process in mammalian cells remains to be uncovered, and
`silencing might occur post-transcriptionally and/or transcription-
`ally. In Drosophila lysate, siRNA duplexes mediate post-transcrip-
`tional gene silencing by reconstitution of siRNA-protein complexes
`(siRNPs), which guide mRNA recognition and targeted cleavage6,7,9.
`In plants, dsRNA-mediated post-transcriptional silencing has also
`been linked to DNA methylation, which may also be directed by 21-
`
`Iamin A/C
`siRNA
`
`GL2 Pp-luc
`siRNA
`
`Buffer
`
`b
`
`e
`
`h
`
`c
`
`f
`
`i
`
`Iamin A/C
`siRNA
`
`GL2 Pp-luc
`siRNA
`
`Buffer
`
`lamin A
`lamin C
`
`vimentin
`
`a
`
`d
`
`g
`
`lamin A/C ab
`
`NuMA ab
`
`Hoechst
`
`j
`
`Mr
`205K
`
`116K
`97K
`66K
`
`55K
`45K
`
`36K
`
`k
`
`55K
`45K
`
`Figure 4 Silencing of nuclear envelope proteins lamin A/C in HeLa cells. Triple
`fluorescence staining of cells transfected with lamin A/C siRNA duplex (a, d, g), with GL2
`luciferase siRNA duplex (nonspecific siRNA control) (b, e, h), and with buffer only (c, f, i).
`a–c, Staining with lamin A/C specific antibody; d–f, staining with NuMA-specific
`antibody; g–i, Hoechst staining of nuclear chromatin. Bright fluorescent nuclei in a
`represent untransfected cells. j, k, Western blots of transfected cells using lamin A/C- ( j)
`or vimentin-specific (k) antibodies. The Western blot was stripped and re-probed with
`vimentin antibody to check for equal loading of total protein.
`
`NATURE | VOL 411 | 24 MAY 2001 | www.nature.com
`
`© 2001 Macmillan Magazines Ltd
`
`497
`
`Benitec - Exhibit 1016 - page 4
`
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`
`letters to nature
`
`immunofluorescence. Monoclonal V9 vimentin-specific antibody was used at 1/2,000
`dilution. For western blotting, transfected cells grown in 24-well plates were trypsinized
`and harvested in SDS sample buffer. Equal amounts of total protein were separated on
`12.5% polyacrylamide gels and transferred to nitrocellulose. Standard immunostaining
`was carried out using ECL enhanced chemiluminescence technique (Amersham Phar-
`macia).
`For immunofluorescence, transfected cells grown on glass coverslips in 24-well plates
`were fixed in methanol for 6 min at -10 8C. Target gene specific and control primary
`antibody were added and incubated for 80 min at 37 8C. After washing in phosphate
`buffered saline (PBS), Alexa 488-conjugated anti-rabbit (Molecular Probes) and Cy3-
`conjugated anti-mouse (Dianova) antibodies were added and incubated for 60 min at
`37 8C. Finally, cells were stained for 4 min at room temperature with Hoechst 33342 (1 mM
`in PBS) and embedded in Mowiol 488 (Hoechst). Pictures were taken using a Zeiss
`Axiophot camera with a Fluar 40/1.30 oil objective and MetaMorph Imaging Software
`(Universal Imaging Corporation) with equal exposure times for the specific antibodies.
`
`Received 20 February; accepted 26 April 2001.
`
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