Previous Article | Next Article 
Molecular and Cellular Biology, November 2001, p. 7807-7816, Vol. 21, No. 22
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.22.7807-7816.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Specific Double-Stranded RNA Interference in
Undifferentiated Mouse Embryonic Stem Cells
Shicheng
Yang,1
Stephen
Tutton,1
Eric
Pierce,2 and
Kyonggeun
Yoon1,*
Department of Dermatology and Cutaneous
Biology and Department of Biochemistry and Molecular Pharmacology,
Jefferson Institute of Molecular Medicine, Thomas Jefferson University,
and Jefferson Medical College,1 and
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye
Institute, University of Pennsylvania School of
Medicine,2 Philadelphia, Pennsylvania
Received 18 April 2001/Returned for modification 4 June
2001/Accepted 16 August 2001
Specific mRNA degradation mediated by double-stranded RNA (dsRNA)
interference (RNAi) is a powerful way of suppressing gene expression in
plants, nematodes, and fungal, insect, and protozoan systems. However,
only a few cases of RNAi have been reported in mammalian systems. Here,
we investigated the feasibility of the RNAi strategy in several
mammalian cells by using the enhanced green fluorescent protein gene as
a target, either by in situ production of dsRNA from transient
transfection of a plasmid harboring a 547-bp inverted repeat or by
direct transfection of dsRNA made by in vitro transcription. Several
mammalian cells including differentiated embryonic stem (ES) cells did
not exhibit specific RNAi in transient transfection. This long dsRNA,
however, was capable of inducing a sequence-specific RNAi for the
episomal and chromosomal target gene in undifferentiated ES cells.
dsRNA at 8.3 nM decreased the cognate gene expression up to 70%.
However, RNAi activity was not permanent because it was more pronounced
in early time points and diminished 5 days after transfection. Thus,
undifferentiated ES cells may lack the interferon response, similar to
mouse embryos and oocytes. Regardless of their apparent RNAi activity,
however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that
mammalian cells may possess RNAi activity but nonspecific activation of
the interferon response by longer dsRNA may mask the specific RNAi. The
findings offer an opportunity to use dsRNA for inhibition of gene
expression in ES cells to study differentiation.
*
Corresponding author. Mailing address: Department of
Dermatology and Cutaneous Biology, Department of Biochemistry and
Molecular Pharmacology, Jefferson Institute of Molecular Medicine,
Thomas Jefferson University, and Jefferson Medical College, 233 South 10th Street, Philadelphia, PA 19107. Phone: (215) 503-5434. Fax: (215)
503-5788. E-mail: kyonggeun.yoon{at}mail.tju.edu.
Molecular and Cellular Biology, November 2001, p. 7807-7816, Vol. 21, No. 22
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.22.7807-7816.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Olejniczak, M., Galka, P., Krzyzosiak, W. J.
(2010). Sequence-non-specific effects of RNA interference triggers and microRNA regulators. Nucleic Acids Res
38: 1-16
[Abstract]
[Full Text]
-
Umbach, J. L., Cullen, B. R.
(2009). The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev.
23: 1151-1164
[Abstract]
[Full Text]
-
Svoboda, P., Stein, P.
(2009). RNAi Experiments in Mouse Oocytes and Early Embryos. CSH Protocols
2009: pdb.top56-pdb.top56
[Abstract]
[Full Text]
-
Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P., Blelloch, R.
(2008). Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev.
22: 2773-2785
[Abstract]
[Full Text]
-
Dinger, M. E, Mercer, T. R, Mattick, J. S
(2008). RNAs as extracellular signaling molecules. J Mol Endocrinol
40: 151-159
[Abstract]
[Full Text]
-
Xu, Y., Mirmalek-Sani, S.-H., Lin, F., Zhang, J., Oreffo, R. O.C.
(2007). Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells. RNA
13: 1179-1183
[Abstract]
[Full Text]
-
Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S., Hannon, G. J.
(2005). Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci. USA
102: 12135-12140
[Abstract]
[Full Text]
-
Yan, W., Ma, L., Stein, P., Pangas, S. A., Burns, K. H., Bai, Y., Schultz, R. M., Matzuk, M. M.
(2005). Mice Deficient in Oocyte-Specific Oligoadenylate Synthetase-Like Protein OAS1D Display Reduced Fertility. Mol. Cell. Biol.
25: 4615-4624
[Abstract]
[Full Text]
-
Zhou, H., Xia, X. G., Xu, Z.
(2005). An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res
33: e62-e62
[Abstract]
[Full Text]
-
Wobus, A. M., Boheler, K. R.
(2005). Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy. Physiol. Rev.
85: 635-678
[Abstract]
[Full Text]
-
O'Shea, K. S.
(2004). Self-renewal vs. Differentiation of Mouse Embryonic Stem Cells. Biol. Reprod.
71: 1755-1765
[Abstract]
[Full Text]
-
Wang, Q., Carmichael, G. G.
(2004). Effects of Length and Location on the Cellular Response to Double-Stranded RNA. Microbiol. Mol. Biol. Rev.
68: 432-452
[Abstract]
[Full Text]
-
Szabo, P. E., Tang, S.-H. E., Silva, F. J., Tsark, W. M. K., Mann, J. R.
(2004). Role of CTCF Binding Sites in the Igf2/H19 Imprinting Control Region. Mol. Cell. Biol.
24: 4791-4800
[Abstract]
[Full Text]
-
Kariko, K., Bhuyan, P., Capodici, J., Weissman, D.
(2004). Small Interfering RNAs Mediate Sequence-Independent Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 3. J. Immunol.
172: 6545-6549
[Abstract]
[Full Text]
-
Yang, S., Cho, Y. S., Chennathukuzhi, V. M., Underkoffler, L. A., Loomes, K., Hecht, N. B.
(2004). Translin-associated Factor X Is Post-transcriptionally Regulated by Its Partner Protein TB-RBP, and Both Are Essential for Normal Cell Proliferation. J. Biol. Chem.
279: 12605-12614
[Abstract]
[Full Text]
-
Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R., Saigo, K.
(2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res
32: 936-948
[Abstract]
[Full Text]
-
Milhavet, O., Gary, D. S., Mattson, M. P.
(2003). RNA Interference in Biology and Medicine. Pharmacol. Rev.
55: 629-648
[Abstract]
[Full Text]
-
Einav, Y., Shistik, E., Shenfeld, M., Simons, A. H., Melton, D. W., Canaani, D.
(2003). Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens. Molecular Cancer Therapeutics
2: 1121-1128
[Abstract]
[Full Text]
-
Opalinska, J. B., Gewirtz, A. M.
(2003). Therapeutic Potential of Antisense Nucleic Acid Molecules. Sci Signal
2003: pe47-pe47
[Abstract]
[Full Text]
-
SAUNDERS, L. R., BARBER, G. N.
(2003). The dsRNA binding protein family: critical roles, diverse cellular functions. FASEB J.
17: 961-983
[Abstract]
[Full Text]
-
Hemmings-Mieszczak, M., Dorn, G., Natt, F. J., Hall, J., Wishart, W. L.
(2003). Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant Rat P2X3 receptor. Nucleic Acids Res
31: 2117-2126
[Abstract]
[Full Text]
-
Kosciolek, B. A., Kalantidis, K., Tabler, M., Rowley, P. T.
(2003). Inhibition of Telomerase Activity in Human Cancer Cells by RNA Interference. Molecular Cancer Therapeutics
2: 209-216
[Abstract]
[Full Text]
-
Capodici, J., Kariko, K., Weissman, D.
(2002). Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference. J. Immunol.
169: 5196-5201
[Abstract]
[Full Text]
-
Yang, D., Buchholz, F., Huang, Z., Goga, A., Chen, C.-Y., Brodsky, F. M., Bishop, J. M.
(2002). Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA
99: 9942-9947
[Abstract]
[Full Text]
-
Tuschl, T., Borkhardt, A.
(2002). Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy. Mol. Interv.
2: 158-167
[Abstract]
[Full Text]
-
Yu, J.-Y., DeRuiter, S. L., Turner, D. L.
(2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA
99: 6047-6052
[Abstract]
[Full Text]
-
Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S.
(2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev.
16: 948-958
[Abstract]
[Full Text]
-
Zhou, Y., Ching, Y.-P., Kok, K. H., Kung, H.-f., Jin, D.-Y.
(2002). Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA. Nucleic Acids Res
30: 1664-1669
[Abstract]
[Full Text]
-
Yu, J.-Y., DeRuiter, S. L., Turner, D. L.
(2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA
99: 6047-6052
[Abstract]
[Full Text]