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Plant and Cell Physiology 2004 45(11):1648-1657; doi:10.1093/pcp/pch186
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© 2004 Oxford University Press

DNA Interference: a Simple and Efficient Gene-Silencing System for High-Throughput Functional Analysis in the Fern Adiantum

Hiroko Kawai-Toyooka1,3, Chihiro Kuramoto1,3, Kyoko Orui1,4, Kentaro Motoyama1, Kazuhiro Kikuchi2, Takeshi Kanegae1 and Masamitsu Wada1,2,5

1 Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, 192-0397 Japan
2 Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Okazaki, 444-8585 Japan

RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach ‘DNA interference’.

3 These authors contributed equally to this work.

4 Present address: Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchuu, Tokyo, 183-8509 Japan

5 Corresponding author: E-mail, wada-masamitsu{at}c.metro-u.ac.jp; Fax, +81-426-77-2559.


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