RESEARCH ARTICLE
`
`4557
`
`Identification of essential genes in cultured
`mammalian cells using small interfering RNAs
`Jens Harborth1,*, Sayda M. Elbashir2,*, Kim Bechert1, Thomas Tuschl2,‡ and Klaus Weber1,‡
`1Department of Biochemistry and Cell Biology and 2Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry,
`Am Fassberg 11, 37077 Göttingen, Germany
`*These authors contributed equally to this work
`‡Authors for correspondence (e-mail: ttuschl@mpibpc.gwdg.de; office.weber@mpibpc.gwdg.de)
`
`Accepted 10 October 2001
`Journal of Cell Science 114, 4557-4565 (2001) ©The Company of Biologists Ltd
`
`SUMMARY
`
`in
`the first RNAi-induced phenotypes
`We report
`mammalian cultured cells using RNA
`interference
`mediated by duplexes of 21-nt RNAs. The 21 gene
`products studied have different functions and subcellular
`localizations. Knockdown experiments monitored by
`immunofluorescence and immunoblotting show that even
`major cellular proteins such as actin and vimentin can be
`silenced efficiently. Genes were classified as essential or
`nonessential depending on impaired cell growth after RNA
`silencing. Phenotypes also involved altered cell morphology
`and aberrant mitotic arrest. Among the essential genes
`identified by RNAi for which such information was
`previously not available are lamin B1, lamin B2, NUP153,
`GAS41, ARC21, cytoplasmic dynein, the protein kinase
`cdk1 and both b - and g -actin. Newly defined nonessential
`genes are emerin and zyxin. Several genes previously
`
`characterized by other methods such as knockout of
`murine genes are included as internal controls and gave
`identical results when RNAi was used. In the case of two
`nonessential genes (lamin A/C and zyxin) RNAi provides a
`recognizable phenotype.
`Our results complete the characterization of the
`mammalian nuclear lamins. While lamins A/C appear as
`nonessential proteins in the mouse embryo and in RNAi
`treated cultured cells, the two other lamins, B1 and B2, are
`now identified as essential proteins. Interestingly the inner
`nuclear membrane protein emerin, thought to be a ligand
`of lamin A/C, is also a nonessential protein in tissue culture
`cells.
`
`Key words: Functional genomics, Gene silencing, Mammalian cells,
`Nuclear lamins, RNA interference
`
`INTRODUCTION
`
`Gene function has been determined traditionally by methods
`such as deletion of murine genes, the introduction of (mutated
`and/or dominant negative)
`transgenes,
`the molecular
`characterization of human hereditary diseases, and targeting of
`genes by antisense techniques (Porter, 1998). Microinjection
`of an antibody into tissue culture cells (Blangy et al., 1995)
`and the use of Xenopus oocyte extracts (Desai et al., 1999;
`Abrieu et al., 2000) can also provide information on protein
`functions. As the sequencing of the human and murine
`genomes approaches completion there is an increasing demand
`for quick, robust and efficient mechanisms to analyse gene
`function in mammalian cell culture.
`RNA interference (RNAi) provides a new approach for
`elucidation of gene function. RNAi is a sequence-specific,
`post-transcriptional gene silencing mechanism initiated in
`animals and plants by the introduction of double stranded RNA
`(dsRNA) homologous in sequence to the silenced gene (Fire,
`1999; Bass, 2000; Cogoni and Macino, 2000; Plasterk and
`Ketting, 2000; Sijen and Kooter, 2000; Hammond et al., 2001;
`Matzke et al., 2001; Sharp, 2001; Tuschl, 2001; Waterhouse et
`al., 2001). RNAi has significantly advanced our understanding
`of gene function in the nematode C. elegans and about one
`third of the nematode genome has already been subjected to
`functional analysis by RNAi (Fraser et al., 2000; Gonczy et al.,
`
`2000; Barstead, 2001; Hope, 2001; Kim, 2001; Maeda et al.,
`2001). Mammalian cells were until recently not amenable to
`RNAi since use of in vitro transcribed, long dsRNAs (>30 bp)
`led to activation of a global, sequence unspecific response
`resulting in blockage of initiation of protein synthesis and
`mRNA degradation (Bass, 2001). Although RNA interference
`seems to work in early mouse development (Svoboda et al.,
`2000; Wianny and Zernicka-Goetz, 2000), it seemed not
`generally applicable to mammalian cells (Caplen et al., 2000;
`Ui-Tei et al., 2000).
`We recently reported that duplexes of 21-nt RNAs with 2-nt
`3¢ overhang, introduced by transfection into human and other
`mammalian cultured cells specifically interfered with gene
`expression and bypassed the sequence independent response of
`mammalian cells to long dsRNA (Elbashir et al., 2001a). These
`short RNA duplexes resemble the processing products from
`long dsRNAs, and are referred to as small interfering RNAs
`(siRNAs) (Elbashir et al., 2001b). One of the enzymes involved
`in processing of long dsRNAs has recently been identified and
`is a member of the RNase III family of nucleases (Bernstein et
`al., 2001).
`We previously documented siRNA-induced silencing of
`lamin A/C in HeLa cells and mentioned that this approach also
`worked for lamin B1 and the nuclear mitotic apparatus protein
`NuMA but not for vimentin. We speculated that the negative
`result with vimentin could be due to the abundance of the
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`protein or the particular RNA duplex used (Elbashir et al.,
`2001a). Here we extend our analysis to a large number of genes
`in mammalian tissue culture cells. We show that RNAi results
`in silencing of major cellular proteins such as actin and that
`vimentin can be effectively silenced by selecting three new
`RNA duplexes. We report the first phenotypes obtained by
`RNAi in mammalian cells. Among the essential genes
`identified are lamin B1, lamin B2, NUP153, GAS41, ARC21,
`cytoplasmic dynein and the mitotic protein kinase cdk1, for
`which such information was not available previously by
`ablation of murine genes or other approaches.
`
`MATERIALS AND METHODS
`
`siRNA preparation
`To design target-specific siRNA duplexes, we selected sequences of
`the type AA(N19)UU (N, any nucleotide) from the open reading frame
`of the targeted mRNA, in order to obtain a 21-nt sense and 21-nt
`antisense strand with symmetric 2-nt 3¢ overhangs of identical
`sequence. We used 2¢ -deoxythymidines instead of uridine residues in
`the 3¢ overhangs to reduce costs of RNA synthesis and to enhance
`nuclease resistance. It may also possible to design siRNAs with other
`sequences in the overhang, or to select siRNAs from the sequences
`AA(N21) by changing the last two nucleotides of the sense siRNA to
`TT (the 2-nt overhang of the sense siRNAs is not believed to
`contribute to target recognition). A selected siRNA sequence was also
`submitted to a BLAST search against the human genome sequence
`to ensure that only one gene of the human genome was targeted.
`21-nt RNAs were chemically synthesized using Expedite RNA
`phosphoramidites
`and
`thymidine phosphoramidite
`(Proligo,
`Hamburg, Germany), deprotected and gel-purified as described
`(Elbashir et al., 2001a; Elbashir et al., 2001b) or purchased from
`Dharmacon (Lafayette, CO) in deprotected and desalted form.
`The accession numbers given below in brackets are from GenBank.
`The siRNA sequence targeting NuMA (Z11583) was from position
`3988-4010 relative to the start codon; GAS41 (NM_006530) 327-349;
`SV40 T-antigen (62000) 26-48, 639-661; lamin A/C (NM_005572)
`608-630; lamin B1 (NM_005573) 672-694; lamin B2 (M94362)
`sequence with missing 5¢ end); LAP2
`1457-1479
`(of
`the
`(NM_003276) 37-59, common region; emerin (NM_000117) 628-
`650; Nup153 (NM_005124) 2593-2615; b -actin (NM_001101) 972-
`994; g -actin (NM_001614) 8-30; ARC21 (AF006086) 181-203;
`mouse zyxin (X99063) 1357-1379; mouse vinculin (L18880) 2923-
`2945; VASP (XM_009141) 1087-1109; vimentin (NM_003380) 346-
`368, 1145-1167, 863-885, 1037-1059; keratin 18 (NM_000224) 1154-
`1176; Eg5 (NM_004523) 1547-1569; CENP-E (NM_001813)
`944-966; cytoplasmic dynein 1 heavy chain (U53530) 509-531 of
`partial cDNA; cdk1 (NM_001786) 125-147. As unspecific siRNA
`control a sequence targeting firefly (Photinus pyralis) luciferase gene
`(X65324) 153-175 was used. siRNA duplex formation (annealing)
`was performed as previously described (Elbashir et al., 2001a).
`
`Cell culture and transfection
`Human HeLa SS6 cells and F5 and FR(wt648) rat fibroblast cells
`(Zerrahn and Deppert, 1993) were grown at 37(cid:176) C in Dulbecco’s
`modified Eagle medium supplemented with 10% FCS, penicillin and
`streptomycin. Cells were regularly passaged to maintain exponential
`growth. The day before transfection, cells were trypsinized, diluted
`with fresh medium without antibiotics and transferred to 24-well
`plates (500 m
`l per well). Transient transfection of siRNAs was carried
`out using Oligofectamine (Life Technologies). 12 m
`l OPTIMEM 1
`medium (Life Technologies) and 3 m
`l Oligofectamine per well were
`preincubated for 5-10 minutes at room temperature. During the time
`for this incubation 50 m
`l OPTIMEM 1 medium were mixed with 3 m
`l
`siRNA. The two mixtures were combined and incubated for 20
`
`minutes at room temperature for complex formation. After addition
`of 32 m
`l of OPTIMEM 1 medium to the mixture, the entire mixture
`was added to the cells in one well resulting in a final concentration of
`100 nM for the siRNAs. The addition of 32 m
`l OPTIMEM 1 medium
`is optional and was only used to adjust the final culture volume to 600
`l. Cells were usually assayed 40-48 hours after transfection, but in
`some cases also after 70 hours. Specific silencing was confirmed by
`at least three independent experiments.
`
`Immunofluorescence microscopy, antibodies and
`immunoblotting
`Immunofluorescence and chromatin staining was performed as
`described (Elbashir et al., 2001a). Pictures were taken using a Zeiss
`Axiophot with a F Fluar 40· /1.30 oil objective and MetaMorph
`Imaging Software (Universal Imaging Corporation, West Chester, PA)
`with equal exposure times for the silenced and the control treated
`cells. For phase-contrast microscopy, cells were mounted in Hepes
`buffered DMEM medium supplemented with 10% FCS. Pictures were
`taken using a Plan-Neofluar 25· /0.8 objective.
`Several antibodies were kindly provided by B. Burke (monoclonal
`Nup153), W. Deppert (monoclonal SV40 T-antigen 108), M. Osborn
`(monoclonal lamin A/C clone 636, NuMA, vimentin monoclonal V9,
`keratin 18 CK2) and J. Wehland (monoclonal ARC21 7H3, VASP
`273D4, vinculin, zyxin 164ID4). Commercial antibodies were from
`Abcam (b -actin), Novocastra (lamin B2 clone LN43.2, emerin clone
`4G5), Santa Cruz Biotechnology (lamin B1 C-20), Sigma (a -tubulin)
`and Transduction Laboratories (LAP2 clone 27). The antibody for
`GAS41 was described previously (Harborth et al., 2000).
`For western blotting, transfected cells grown in 24-well plates, were
`trypsinized, washed once in ice-cold PBS and harvested. Cells from
`one well were solubilized in 50 m
`l SDS sample buffer and boiled for
`5 minutes. Equal amounts of total protein were separated on 7.5
`or 12.5% polyacrylamide gels and transferred to nitrocellulose.
`Immunostaining with specific antibodies and peroxidase-conjugated
`secondary antibodies (Dako, Denmark) diluted 1:20,000 was carried
`out using the ECL technique (Amersham Pharmacia Biotech). To
`confirm equal loading, blots were stripped (Re-Blot Western Blot
`Recycling Kit, Chemicon) and reprobed with the vimentin V9
`antibody or in case of vimentin silencing with the b -actin antibody.
`
`RESULTS
`
`Selection of target genes
`We examined silencing of 21 genes expressed in cultured
`mammalian cells by RNA interference with duplexes of 21-nt
`RNAs (Table 1). These included 18 genes in human HeLa cells,
`1 gene in SV40-transformed rat fibroblasts, and 2 genes in
`mouse 3T3 cells. The sequences of the siRNA duplexes were
`selected from the coding region of the target mRNAs as
`described in Materials and Methods. In all transfection
`experiments cultures were transfected in parallel with target-
`specific siRNA duplexes and a nonspecific duplex of firefly
`luciferase from Photinus pyralis (GL2) at the same final
`concentration as the specific siRNAs tested (Elbashir et al.,
`2001a). As a further control we used the addition of buffer
`instead of the volume of siRNA added to the culture.
`Gene silencing was documented by immunoblotting and
`immunofluorescence, except in five cases where no suitable
`antibodies were available. To screen for phenotypes, we looked
`for impaired cell growth and altered cell morphology
`monitored by phase microscopy. Aberrant mitotic arrest was
`visualized using the a -tubulin antibody.
`The genes selected for this study code for a wide range of
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`Table 1. Gene silencing by RNAi: growth arrest and phenotypes in cell culture
`Silencing*
`Essential
`Phenotypic change
`Gene function by other methods
`
`RNAi in mammalian cells
`
`4559
`
`+
`
`+
`+
`
`+
`+
`+
`+
`+
`+
`
`+
`nd
`+
`+
`+
`+
`+
`+
`
`nd
`
`nd
`
`nd
`nd
`
`+
`
`+
`–
`
`–
`+
`+
`–
`–
`+
`
`+
`+
`+
`–
`+
`–
`–
`–
`
`+
`
`+
`
`+
`+
`
`Apoptotic
`
`Arrest
`
`Emerin displaced
`Apoptotic
`Apoptotic
`
`Blebbing
`Blebbing
`
`Loss of stress fibers
`
`Aberrant mitotic, monopolar spindles
`
`Aberrant mitotic, multipolar spindles
`
`Aberrant mitotic arrest
`Premitotic arrest
`
`Antibody injection, mutant cDNAs: essential
`for mitosis and nuclear formation
`
`Null mice: nonessential in development;
`essential after birth
`
`Null mice: embryonic lethal
`
`Null mice: nonessential
`Null mice: nonessential
`
`Antibody injection, inhibitor: essential for
`centrosome separation
`Antisense and immunodepletion: essential
`kinetochore-associated motor
`
`Gene
`Nuclear proteins
`NuMA
`
`GAS41
`SV40 T-antigen
`Nuclear envelope proteins
`Lamin A/C
`Lamin B1
`Lamin B2
`LAP2
`Emerin
`Nup153
`Cytoskeletal cytoplasmic proteins
`b Actin
`g Actin
`ARC21
`VASP
`Vinculin
`Zyxin
`Vimentin
`Keratin 18
`Mitotic proteins
`Eg5
`
`CENP-E
`
`Cytoplasmic dynein
`Cdk1
`
`*Gene silencing was monitored by immunofluorescence and/or immunoblotting; nd, not determined because of lack of a suitable antibody. Silencing was
`performed in human HeLa cells, except for SV40 T antigen (rat fibroblasts) and vinculin and zyxin (mouse 3T3). A stop in growth was taken to indicate that the
`protein is essential. For gene function by other methods see text.
`
`Fig. 1. Silencing of SV40 large T
`antigen in a stably transformed rat
`fibroblast cell line (F5). Triple
`fluorescence staining of cells
`transfected with T-antigen siRNA
`duplex (A,C,E), and with GL2
`luciferase siRNA duplex serving as
`control (B,D,F). (A,B) Staining with
`T-antigen specific antibody,
`(C,D) staining with NuMA specific
`antibody, (E,F) Hoechst staining of
`chromatin. The few cells cells in A
`that are stained for T antigen were
`probably nontransfected.
`(G) Western blot of cells transfected
`with T antigen siRNA (left) or
`luciferase siRNA (right) probed
`with T antigen-specific antibody.
`The blot was stripped and reprobed
`with vimentin antibody (bottom).
`Magnification 480·
`(A-F).
`
`Benitec - Exhibit 1012 - page 3
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`

`
`Fig. 2. Silencing of lamin A/C in HeLa cells results in displacement
`of emerin. Cells transfected with lamin A/C siRNA (A) and with
`luciferase siRNA (B) were stained with emerin antibody. Cells with
`reduced lamin A/C expression (A) show enrichment of emerin in the
`cytoplasm. Magnification 480·
`.
`
`with the observation that mice null for A/C show normal
`development and only die one to two months after birth due to
`muscular dystrophy (Sullivan et al., 1999). Also, lamin A/C is
`acquired only late in development (Röber et al., 1989). Despite
`the absence of a growth defect, lamin A/C knockdown cells
`show a distinct phenotype. Similar to fibroblasts derived from
`embryos of lamin A/C null mice (Sullivan et al., 1999), lamin
`A/C silenced HeLa cells show an unusual distribution of the
`inner nuclear membrane protein emerin (Fig. 2). The
`cytoplasmic distribution of emerin probably within the
`endoplasmic reticulum is strongly enhanced and supports the
`view that lamin A/C may serve to immobilize emerin.
`Therefore, knockdown of genes that are not required for cell
`growth may still cause distinct phenotypes in cultured cells,
`which can be monitored by appropriate means.
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`proteins distributed in different cellular compartments. The
`study includes three nuclear proteins, six proteins of the
`nuclear envelope, eight cytoplasmic cytoskeletal proteins, three
`microtubular motor proteins and the protein kinase cdk1.
`Seven genes, which were already characterized by other
`methods such as murine knockouts, microinjection of
`antibodies and transfection by mutant cDNAs served as
`controls. In these cases, RNAi mediated by siRNAs provided
`the same result as the earlier studies (Table 1).
`
`Knockdown of nuclear proteins
`Antibody microinjection and transfection with mutant cDNAs
`showed that the nuclear mitotic apparatus protein NuMA is
`essential for normal spindles and reformation of nuclei and that
`NuMA is involved in the early stages of apoptosis (Gueth-
`Hallonet et al., 1999). Consistent with these results we
`observed siRNA-induced growth arrest and detected apoptotic
`cells (Table 1). NuMA was shown to interact with GAS41, a
`poorly characterized, but highly conserved protein
`in
`eukaryotic nuclei (Harborth et al., 2000). Targeting of GAS41
`causes growth arrest of HeLa cells, demonstrating for the first
`time that GAS41 is an essential protein.
`As another example of a nuclear protein we tried to target
`SV40 large T antigen in transformed rat fibroblasts using
`two duplexes. Interestingly only one of them resulted in
`specific silencing (see also vimentin) as demonstrated by
`immunofluorescence and western blotting (Fig. 1). At least
`under the conditions used cells continued to grow. It remains
`to be seen whether this is due to the residual amount of antigen
`still present or whether T antigen is indeed nonessential for
`growth.
`
`Knockdown of proteins of the
`nuclear envelope
`The nuclear envelope consists of a
`double membrane, pores, and the
`lamina, a filamentous meshwork
`located between the inner nuclear
`membrane
`and
`the
`peripheral
`chromatin. We silenced the nuclear
`lamins A/C, B1 and B2, the lamina-
`associated protein LAP2, the inner
`nuclear membrane protein emerin,
`and
`the nuclear pore component
`Nup153. Knockdown of lamin A/C in
`HeLa cells did not affect cell growth
`(Elbashir et al., 2001a), consistent
`
`Fig. 3. Silencing of lamin B1 and lamin
`B2. HeLa cells were transfected with
`lamin B1 siRNA (A,C), lamin B2 siRNA
`(E) or with luciferase siRNA (B,D,F).
`Cells were either double stained with
`lamin B1-specific antibody (A,B) and
`NuMA specific antibody (C,D) or stained
`for lamin B2 (E,F). (G) Western blot of
`cells transfected with lamin B1 (left) or
`luciferase (right) siRNA duplexes using
`lamin B1 antibody (top). The blot was
`stripped and re-probed with vimentin
`antibody to check for equal loading
`(bottom). Magnification 480·
`(A-F).
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`RNAi in mammalian cells
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`4561
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`Fig. 4. Silencing of the nuclear pore complex protein Nup153.
`(A) Phase-contrast of HeLa cells 3 days after transfection with
`Nup153 siRNA. (B) Western blot of cells transfected with Nup153
`siRNA (left) or with luciferase siRNA (right) probed with Nup153-
`specific antibody. Magnification 300·
`(A).
`
`is
`lamin A/C, whose expression
`to
`In contrast
`developmentally regulated, lamin B1 and lamin B2 are
`constitutively expressed (Röber et al., 1989). Lamin B1 and
`lamin B2 were efficiently silenced 40 hours post-transfection as
`shown by immunofluorescence or immunoblot (Fig. 3). Later,
`cells stop growing and become apoptotic. We demonstrated by
`siRNA-mediated silencing that both B lamins are essential
`proteins (Table 1). The observed cessation of cell growth upon
`siRNA transfection indicates that both B-type lamins are
`necessary to establish a normal nuclear lamina in HeLa cells.
`Apoptosis induced by lamin B1 and B2 silencing is consistent
`with the previous finding that perturbation of post-mitotic lamin
`B1 assembly leads to apoptosis (Burke, 2001). Interestingly
`transfection with a lamin B1 cDNA carrying a large deletion in
`the central rod domain leads to lobulated nuclei and growth
`arrest (Schirmer et al., 2001). These observations do not rule
`out additional functions for B-type lamins in DNA replication
`and transcription (Wilson et al., 2001).
`The siRNA duplex directed against the nuclear envelope
`protein LAP2 had no effect on cell growth, yet an effect may
`have been expected because of the known interaction with the
`essential B-type lamins (Burke, 2001; Hutchison et al., 2001).
`Although the LAP2 protein levels were significantly reduced
`(Table 1), it is unclear whether the residual protein may be
`responsible for the absence of an effect. Similarly, silencing of
`the inner nuclear membrane protein emerin, had no effect in
`HeLa cells. Although lamin A/C knockdown affected emerin
`localization (see above) (Sullivan et al., 1999), the reverse
`effect was not observed, and silencing of emerin did not
`influence the nuclear lamin A/C staining pattern.
`Nup153 is a protein of the nuclear pore complex and belongs
`to the F/GXFG family of nuclear pore proteins (Radu et al.,
`1995). HeLa cells transfected with siRNAs directed against
`Nup153 rounded up and showed growth arrest (Fig. 4),
`indicating that this nuclear pore protein is essential.
`
`Knockdown of cytoskeletal proteins
`We previously reported a problem with silencing of vimentin
`in HeLa cells (Elbashir et al., 2001a) and speculated that this
`was either due to the abundance of the protein or perhaps due
`to the sequence selected for siRNA synthesis. We first
`addressed the issue of abundance and tested siRNAs directed
`against other major proteins expressed in HeLa cells. Silencing
`of b -cytoplasmic actin was readily achieved as monitored by a
`specific antibody (Fig. 5). Reduction of b -actin levels led to a
`
`Fig. 5. Silencing of cytoplasmic actins in HeLa cells. (A) Phase-
`contrast of cells transfected with b -actin siRNA duplex. Transfected
`cells show blebbing. (B) Western blot of cells transfected with b -
`actin (left) or luciferase (right) siRNA duplexes using b -actin
`antibody. The blot was stripped and re-probed with vimentin
`antibody to check for equal loading. (C) Phase-contrast of cells
`transfected with g -actin siRNA duplex. Transfected cells show a
`blebbing phenotype similar to cells with b -actin knockdown.
`Magnification 480·
`(A,C).
`
`stop in cell growth and phase microscopy revealed very strong
`cellular blebbing. The same phenotype was observed after
`targeting of g -cytoplasmic actin, although the precise g -actin-
`levels could not be measured due to a lack of g -specific
`antibody. These results demonstrate that the expression of
`major cellular proteins can indeed be suppressed by RNAi.
`Therefore, we tested again for silencing of vimentin with three
`newly synthesized duplexes directed against different regions
`of the vimentin mRNA. All newly selected siRNA duplexes
`were effective in silencing vimentin expression as monitored
`by immunoblotting. Seventy hours post-transfection, few
`filaments, which were often fragmented, were detectable
`by immunofluorescence microscopy (Fig. 6). Some cells
`contained only short and thin structures, similar to the vimentin
`squiggles frequently visible in live cells expressing GFP-
`tagged vimentin (Yoon et al., 1998). The absence of a
`characteristic phenotype after silencing was expected because
`vimentin null mice develop and reproduce normally (Colucci-
`Guyon et al., 1994). Knockdown cells for another intermediate
`filament protein, keratin 18, behaved similar to vimentin
`knockdown cells, and keratin 18 knockout mice also appear
`normal (Hesse et al., 2000).
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`Fig. 6. Silencing of the cytoskeletal intermediate filament protein
`vimentin. Cells were transfected with vimentin duplex 2 (bp 1145-
`1167) (A-C) or with GL2 luciferase duplex (D). (A,B,D) Staining
`with vimentin V9 antibody; (C) Hoechst staining. Note the strong
`decrease of vimentin staining in cells transfected with vimentin
`duplex 2 (A) when compared with the control (D). Only at longer
`exposure times (B) can short filamentous structures (vimentin
`squiggles) (arrow) be seen. (E) Western blot using vimentin antibody
`(top panel), and b -actin antibody as control (bottom panel).
`Magnification 460·
`(A-D).
`
`We then targeted three actin-associated proteins, vinculin,
`zyxin and VASP, which are usually found in focal contacts
`(Drees et al., 1999; Critchley, 2000). Successful silencing was
`documented by immunofluorescence and immunoblotting.
`Vinculin is essential in mouse 3T3 fibroblasts. Cells became
`strongly contracted and some cells rounded up and were lost
`from the substratum. Silencing of zyxin in 3T3 cells is far less
`dramatic. The cells stayed flat but actin staining with
`rhodamine phalloidin showed that stress fibers were greatly
`reduced and focal contacts were diminished. Our vinculin
`results fit the observation of embryonic lethality in mouse
`knockout experiments (Xu et al., 1998). VASP is not an
`essential protein in HeLa cells and mice null for VASP are
`normal except for a platelet defect (Aszódi et al., 1999).
`ARC21 is a component of the ARP2/3 complex, which
`regulates actin filament network formation (Higgs and Pollard,
`2001). Using siRNA-mediated silencing we found that ARC21
`is an essential protein in HeLa cells (Table 1).
`
`Knockdown of motor proteins
`RNAi was also used to knockdown three microtubule-
`dependent motor proteins (Table 1). The kinesin related motor
`protein Eg5 was shown earlier to be involved in centrosome
`
`Fig. 7. Silencing of the kinesin-related motor protein Eg5 and of the
`microtubular motor CENP-E. HeLa cells were transfected with Eg5
`siRNA (A,D,G-I) or with luciferase control siRNA (B,C,E,F). Cells
`are stained for a -tubulin (A-C) with a corresponding Hoechst stain
`(D-F). Cells transfected with Eg5 siRNA are arrested in mitosis and
`show monoastral microtubular arrays (A,D). By contrast, control cells
`show normal bipolar spindles in mitosis (B,E) and microtubule
`networks in interphase (C,F). A higher magnification of cells
`transfected with the Eg5 siRNA clearly shows the monopolar spindle
`(G), which does not overlap with the chromatin staining (H); for
`overlay see I. HeLa cells were transfected with CENP-E siRNA (J-M).
`Cells were stained with a -tubulin antibody (J,K) and Hoechst (L,M).
`Note the production of multipolar arrays. The higher magnification of
`a metaphase cell (K,M) indicates that the loss of CENP-E affects
`chromosome congression to the spindle equator (arrows).
`Magnification 480·
`(A-F,J-M); 750·
`(G-I).
`
`separation by antibody microinjection experiments (Blangy et
`al., 1995) and by the use of the specific inhibitor monastrol
`(Mayer et al., 1999). Targeting of Eg5 by siRNA duplexes
`
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`
`RNAi in mammalian cells
`
`4563
`
`inexpensive assay of mammalian gene function. Of the 21
`genes studied, 13 were shown to be essential for cell growth.
`For those genes that were previously analyzed for their specific
`function by other gene targeting methods we find that siRNA-
`induced gene silencing produced equivalent phenotypes (Table
`1).
`Our RNAi results on HeLa cells extend the previous
`characterization of lamin A/C to the other two lamins, B1 and
`B2. Although lamin A/C is not an essential protein in the
`mouse embryo (Sullivan et al., 1999) and in RNAi silenced
`HeLa cells, the two human B type lamins are now established
`by RNAi as essential proteins. Interestingly, in the nematode
`C. elegans there is only a single lamin gene. It encodes a B
`type lamin that is essential for early embryonic development
`(Fraser et al., 2000; Liu et al., 2000). Emerin, an inner nuclear
`membrane protein, is thought to interact with lamin A/C
`(Sullivan et al., 1999) (Fig. 2). We now observe by RNAi that
`emerin like lamin A/C is a nonessential gene in cultured cells.
`After birth both proteins become essential at least in certain
`tissues. Mice lacking lamin A/C die after 4-8 weeks due to
`muscular dystrophy (Sullivan et al., 1999) and three hereditary
`human diseases (Emery-Dreifuss muscular dystrophy, dilated
`cardiomyopathy and familial partial
`lipodystrophy) are
`connected with missense mutations in lamin A/C (Hutchison
`et al., 2001; Wilson et al., 2001). Emerin, by contrast, is
`implicated in X-linked Emery-Dreifuss muscular dystrophy
`(Blone et al., 1994).
`During this study, we experienced in two cases a problem
`that affected the efficiency of gene silencing. For vimentin and
`T antigen we found that the first RNA duplex tested was
`ineffective, yet already the second duplex directed against a
`different region of the target resulted in gene silencing.
`Inspection of the sequences of the ineffective siRNA duplexes
`did not reveal any unusual feature. These two cases illustrate
`the value of an antibody to monitor silencing, particularly
`when no phenotype is observed. Currently we do not know
`whether the occasional ineffectiveness of an RNAi duplex
`arises from a local secondary structure of the mRNA,
`protection of the mRNA by a binding protein, or an as yet
`unidentified
`feature
`in
`the sequence of
`the duplex.
`Alternatively, a minor error in the cDNA sequence or a
`polymorphism has to be considered, as already a single
`base change will render the duplex ineffective (S.M.E.,
`unpublished). Since major cellular proteins such as actin,
`vimentin and keratins can be efficiently silenced in HeLa cells,
`difficulties for siRNA-mediated gene silencing are only
`expected when targeting proteins with an unusually long half-
`life. If the targeted proteins have enzymatic activity rather than
`structural functions, phenotypes may be more difficult to
`identify, since siRNA-based technology only provides a
`knock-down of the targeted protein and not a knockout.
`However, we note that at least in the case of the cyclin-
`dependent protein kinase, cdk1, we readily obtained a
`premitotic cellular arrest. We also foresee some difficulties
`when special cell types are used that are difficult to transfect.
`In HeLa and mouse 3T3 cells as well as rat fibroblasts, we
`reached transfection efficiencies near 90%, but certain other
`cell lines may perform poorly in transfection assays. Here
`other delivery methods for the RNA duplexes such as
`electroporation and microinjection can be explored. Given the
`high intracellular stability of siRNA duplexes, we expect that
`
`Fig. 8. Silencing of the cyclin-dependent kinase 1 (cdk1). HeLa cells
`were transfected with cdk1 siRNA (A,C,D) or with luciferase siRNA
`(B) and stained for lamin A/C (A-C). Cells transfected with cdk1
`siRNA round up. Higher magnification shows that the lamina of
`these cells starts to depolymerize (C). (D) Phase-contrast of HeLa
`cells 2 days after transfection with cdk1 siRNA. Magnification 480·
`(A,B); 760·
`(C); 300·
`(D).
`
`revealed the formation of half spindles in about 40% of
`the arrested cells (Fig. 7) as seen in the previous studies.
`Because an identical phenotype was observed with respect to
`previous studies, we did not document Eg5 silencing by
`immunoblotting. Likewise, the kinetochore-associated motor
`protein CENP-E was shown to be an essential component of
`the mitotic spindle, in agreement with recent studies using
`either antisense-mediated reduction in HeLa cells (Yao et al.,
`2000) or immunodepletion in Xenopus oocyte extracts (Abrieu
`et al., 2000). Because of the characteristic aberrant mitotic
`figures (Fig. 7) we did not attempt to obtain an antibody for
`immunoblots. Finally, RNAi of cytoplasmic dynein, a protein
`thought to act in microtubule-kinetochore interactions (Nigg,
`2001), also resulted in aberrant mitotic arrest (Table 1).
`
`Knockdown of a mitotic kinase
`The last protein included in our analysis of siRNA-induced
`phenotypes was the protein kinase cdk1, which is thought to
`control the M phase of the cell cycle (Nigg, 2001). In contrast
`to the mitotic motor proteins (see above), the knockdown cells
`were arrested prior to spindle formation. This is also visible by
`staining for lamin A/C, which revealed a thin nuclear rim
`pattern (Fig. 8). Since phosphorylation of lamins by the cyclin-
`dependent kinase cdk1 is thought to lead to the complete
`disassembly of the lamina (Nigg, 2001) we assume that the
`partial loss of the lamina observed arises from the reduced
`amount of cdk1 still present after RNA interference.
`
`DISCUSSION
`
`The results of our gene targeting experiments by RNAi with
`21-nt RNAs in mammalian tissue culture cells document the
`general applicability of this simple, fast and relatively
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`4564
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`JOURNAL OF CELL SCIENCE 114 (24)
`
`RNAi can probably be used in organotypic cultures such as
`polarized epithelia and muscle cultures, which need a few
`days to establish. Further improvements of the transfection
`efficiencies in the future may provide silenced cultures that
`are suitable as starting material for biochemical analysis.
`Our main criteria to identify novel phenotypes after gene
`silencing were cell growth a