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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.


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