Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

doi:10.1128/MCB.21.22.7807-7816.2001

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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 geneexpression in plants, nematodes, and fungal, insect, and protozoansystems. However, only a few cases of RNAi have been reportedin mammalian systems. Here, we investigated the feasibility ofthe RNAi strategy in several mammalian cells by using the enhancedgreen fluorescent protein gene as a target, either by in situproduction of dsRNA from transient transfection of a plasmid harboringa 547-bp inverted repeat or by direct transfection of dsRNA madeby in vitro transcription. Several mammalian cells including differentiatedembryonic stem (ES) cells did not exhibit specific RNAi in transienttransfection. This long dsRNA, however, was capable of inducinga sequence-specific RNAi for the episomal and chromosomal targetgene in undifferentiated ES cells. dsRNA at 8.3 nM decreased thecognate gene expression up to 70%. However, RNAi activity wasnot permanent because it was more pronounced in early time pointsand diminished 5 days after transfection. Thus, undifferentiatedES cells may lack the interferon response, similar to mouse embryosand oocytes. Regardless of their apparent RNAi activity, however,cytoplasmic extracts from mammalian cells produced a small RNAof 21 to 22 nucleotides from the long dsRNA. Our results suggestthat mammalian cells may possess RNAi activity but nonspecificactivation of the interferon response by longer dsRNA may maskthe specific RNAi. The findings offer an opportunity to use dsRNAfor inhibition of gene expression in ES cells to studydifferentiation.

^* Corresponding author. Mailing address: Department of Dermatology and Cutaneous Biology, Department of Biochemistry and MolecularPharmacology, Jefferson Institute of Molecular Medicine, ThomasJefferson University, and Jefferson Medical College, 233 South10th Street, Philadelphia, PA 19107. Phone: (215) 503-5434. Fax:(215) 503-5788. E-mail: kyonggeun.yoon{at}mail.tju.edu.

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