1 Department of Biochemistry and Cell Biology and
2 Department 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{at}mpibpc.gwdg.de; office.weber{at}mpibpc.gwdg.de)
Accepted October 10, 2001
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SUMMARY |
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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
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INTRODUCTION |
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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 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.
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MATERIALS AND METHODS |
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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) 1457-1479 (of the sequence with missing 5'end); LAP2 (NM_003276) 37-59, common region; emerin (NM_000117) 628-650; Nup153 (NM_005124) 2593-2615; ß-actin (NM_001101) 972-994; -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°C in Dulbeccos 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 µl per well). Transient transfection of siRNAs was carried out using Oligofectamine (Life Technologies). 12 µl OPTIMEM 1 medium (Life Technologies) and 3 µl Oligofectamine per well were preincubated for 5-10 minutes at room temperature. During the time for this incubation 50 µl OPTIMEM 1 medium were mixed with 3 µl siRNA. The two mixtures were combined and incubated for 20 minutes at room temperature for complex formation. After addition of 32 µl 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 µl OPTIMEM 1 medium is optional and was only used to adjust the final culture volume to 600 µl. 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 40x/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 25x/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 (ß-actin), Novocastra (lamin B2 clone LN43.2, emerin clone 4G5), Santa Cruz Biotechnology (lamin B1 C-20), Sigma (-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 µl 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 ß-actin antibody.
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RESULTS |
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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.
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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.
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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 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 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).
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DISCUSSION |
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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 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 and light or immunofluorescence microscopy, but in some cases, video microscopy and electron microscopy can be expected to contribute valuable information. Sometimes, additional experiments may document a phenotype even when a nonessential gene is targeted. Thus, silencing of zyxin results in a loss of actin stress fibers and cells lacking lamin A/C show misplacement of emerin.
The approach described here is suitable for genome wide analysis of gene function as well as target validation of potentially therapeutic valuable genes. In combination with modern screening methods, one can readily envision high throughput procedures based on siRNAs as a systematic approach to functional genomics of cultured mammalian cells. Based on our results, we anticipate that siRNAs will become a widely used tool for cell biologists to study mammalian gene function, and siRNAs will be particularly useful for the analysis of general cell biological mechanisms such as mitosis, processing and traffic of RNA transcripts, the formation of cellular junctions and membrane trafficking.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Abrieu, A., Kahana, J. A., Wood, K. W. and Cleveland, D. W. (2000). CENP-E as an essential component of the mitotic checkpoint in vitro. Cell, 102, 817-826.[Medline]
Aszódi, A., Pfeifer, A., Ahmad, M., Glauner, M., Zhou, X. H., Ny, L., Andersson, K. E., Kehrel, B., Offermanns, S. and Fässler, R. (1999). The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. EMBO J. 18, 37-48.
Barstead, R. (2001). Genome-wide RNAi. Curr. Opin. Chem. Biol. 5, 63-66.[Medline]
Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell, 101, 235-238.[Medline]
Bass, B. L. (2001). RNA interference, the short answer. Nature 411, 428-429.[Medline]
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.[Medline]
Blangy, A., Lane, H. A., dHérin, P., Harper, M., Kress, M. and Nigg, E. A. (1995). Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83, 1159-1169.[Medline]
Blone, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G. and Toniolo, D. (1994). Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy. Nat. Genet. 8, 323-327.[Medline]
Burke, B. (2001). Lamins and apoptosis: a two-way street? J. Cell Biol. 153, F5-F7.
Caplen, N. J., Fleenor, J., Fire, A. and Morgan, R. A. (2000). dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252, 95-105.[Medline]
Cogoni, C. and Macino, G. (2000). Post-transcriptional gene silencing across kingdoms. Curr. Opin. Genet. Dev. 10, 638-643.[Medline]
Colucci-Guyon, E., Portier, M. M., Dunia, I., Paulin, D., Pournin, S. and Babinet, C. (1994). Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell, 79, 679-694.[Medline]
Critchley, D. R. (2000). Focal adhesions the cytoskeletal connection. Curr. Opin. Cell Biol. 12, 133-139.[Medline]
Desai, A., Murray, A., Mitchison, T. J. and Walczak, C. E. (1999). The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61, 385-412.[Medline]
Drees, B. E., Andrews, K. M. and Beckerle, M. C. (1999). Molecular dissection of zyxin function reveals its involvement in cell motility. J. Cell Biol. 147, 1549-1560.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411, 494-498.[Medline]
Elbashir, S. M., Lendeckel, W. and Tuschl, T. (2001b). RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188-200.
Fire, A. (1999). RNA-triggered gene silencing. Trends Genet. 15, 358-363.[Medline]
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C-elegans chromosome I by systematic RNA interference. Nature 408, 325-330.[Medline]
Gonczy, P., Echeverri, C., Oegema, K., Coulson, A., Jones, S. J. M., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C-elegans using RNAi of genes on chromosome III. Nature 408, 331-336.[Medline]
Gueth-Hallonet, C., Osborn, M. and Compton, D. A. (1999). NuMA. In Guidebook to the Cytoskeletal and Motor Proteins (ed. T. Kreis, and R. Vale), pp. 265-268. Oxford: Oxford University Press.
Hammond, S. M., Caudy, A. A. and Hannon, G. J. (2001). Post-transcriptional gene silencing by double-stranded RNA. Nat. Rev. Genet. 2, 110-119.[Medline]
Harborth, J., Weber, K. and Osborn, M. (2000). GAS41, a highly conserved protein in eukaryotic nuclei, binds to NuMA. J. Biol. Chem. 275, 31979-31985.
Hesse, M., Franz, T., Tamai, Y., Taketo, M. M. and Magin, T. M. (2000). Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality. EMBO J. 19, 5060-5070.
Higgs, H. N. and Pollard, T. D. (2001). Regulation of actin filament network formation through ARP 2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem. 70, 649-676.[Medline]
Hope, I. A. (2001). Broadcast interference functional genomics. Trends Genet. 17, 297-299.[Medline]
Hutchison, C. J., Alvarez-Reyes, M. and Vaughan, O. A. (2001). Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes? J. Cell Sci. 114, 9-19.
Kim, S. K. (2001). Functional genomics: the worm scores a knockout. Curr. Biol. 11, R85-R87.[Medline]
Liu, J., Ben-Shahar, T. R., Riemer, D., Treinin, M., Spann, P., Weber, K., Fire, A. and Gruenbaum, Y. (2000). Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol. Biol. Cell, 11, 3937-3947.
Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. (2001). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11, 171-176.[Medline]
Matzke, M. A., Matzke, A. J. M., Pruss, G. J. and Vance, V. B. (2001). RNA-based silencing strategies in plants. Curr. Opin. Genet. Dev. 11, 221-227.[Medline]
Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R. W., Schreiber, S. L. and Mitchison, T. J. (1999). Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971-974.
Nigg, E. A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21-32.[Medline]
Plasterk, R. H. and Ketting, R. F. (2000). The silence of the genes. Curr. Opin. Genet. Dev. 10, 562-567.[Medline]
Porter, A. (1998). Controlling your losses: conditional gene silencing in mammals. Trends Genet. 14, 73-79.[Medline]
Radu, A., Blobel, G. and Moore, M. S. (1995). Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc. Natl. Acad. Sci. USA, 92, 1769-1773.[Abstract]
Röber, R. A., Weber, K. and Osborn, M. (1989). Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal; a developmental study. Development 105, 365-378.[Abstract]
Schirmer, E. C., Guan, T. L. and Gerace, L. (2001). Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization. J. Cell Biol. 153, 479-489.
Sharp, P. A. (2001). RNA interference 2001. Genes Dev. 15, 485-490.
Sijen, T. and Kooter, J. M. (2000). Post-transcriptional gene-silencing: RNAs on the attack or on the defense? BioEssays, 22, 520-531.[Medline]
Sullivan, T., Escalante-Alcalde, D., Bhatt, H., Anver, M., Bhat, N., Nagashima, K., Stewart, C. L. and Burke, B. (1999). Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147, 913-919.
Svoboda, P., Stain, P., Hayashi, H. and Schultz, R. M. (2000). Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127, 4147-4156.
Tuschl, T. (2001). RNA interference and small interfering RNAs. ChemBioChem 2, 239-245.[Medline]
Ui-Tei, K., Zenno, S., Miyata, Y. and Saigo, K. (2000). Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett. 479, 79-82.[Medline]
Waterhouse, P. M., Wang, M. B. and Lough, T. (2001). Gene silencing as an adaptive defence against viruses. Nature 411, 834-842.[Medline]
Wianny, F. and Zernicka-Goetz, M. (2000). Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2, 70-75.[Medline]
Wilson, K. L., Zastrow, M. S. and Lee, K. K. (2001). Lamins and disease: insights into nuclear infrastructure. Cell 104, 647-650.[Medline]
Xu, W. M., Baribault, H. and Adamson, E. D. (1998). Vinculin knockout results in heart and brain defects during embryonic development. Development 125, 327-337.
Yao, X. B., Abrieu, A., Zheng, Y., Sullivan, K. F. and Cleveland, D. W. (2000). CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint. Nat. Cell Biol. 2, 484-491.[Medline]
Yoon, M., Moir, R. D., Prahlad, V. and Goldman, R. D. (1998). Motile properties of vimentin intermediate filament networks in living cells. J. Cell Biol. 143, 147-157.
Zerrahn, J. and Deppert, W. (1993). Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen. J. Virol. 67, 1555-1563.[Abstract]