Establishment of Evaluation Method for siRNA Delivery Using Stable Cell Line Carrying the Luciferase Reporter Gene
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- Iijima Ayako
- Drug Metabolism & Physicochemistry Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd.
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- Hachisu Rei
- Hokkaido System Science Co., Ltd.
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- Kobayashi Hideo
- Drug Metabolism & Physicochemistry Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd.
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- Hashimoto Kouichi
- Drug Metabolism & Physicochemistry Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd.
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- Asano Daigo
- Drug Metabolism & Physicochemistry Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd.
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- Kikuchi Hiroshi
- Drug Metabolism & Physicochemistry Research Laboratory, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd.
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We determined the influence of siRNA (short interfering RNA) for expression of plasmid DNA (pDNA), when mismatched siRNA and pDNA encoding β-galactosidase (β-gal) were transfected into HeLa cells by the cotransfection method in which they were simultaneously added to the cells. Cationic liposomes (Lipofectamine2000) were used as a gene transfection reagent. The knockdown effect on β-gal was observed even when mismatched siRNA was used, and the effect depended on the amount of added mismatched siRNA. But, there was not a distinct difference of introduction of pDNA into cells between using mismatched siRNA and without using it. We considered that the cotransfection method should be avoided when we confirm RNAi efficiency. The reliable evaluation method for siRNA delivery in vitro was thus established by using NFAT reporter HeLa stable cell line or CHO (pMAM-luc) cell line that had DNA encoding luciferase. The following experimental conditions for each cell line were optimized: cell numbers seeded, total incubation times, concentrations of added inducers, and incubation times after addition of inducers. Transfection performance was compared for six commercially available reagents by this method. No commercially available transfection reagent, however, could reduce luciferase activity by less than one tenth without causing cellular cytotoxicity. Development of novel reagents providing higher transfection effects without cytotoxicity is needed.
収録刊行物
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- Biological & Pharmaceutical Bulletin
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Biological & Pharmaceutical Bulletin 30 (10), 1844-1850, 2007
公益社団法人 日本薬学会
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詳細情報 詳細情報について
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- CRID
- 1390001204624367104
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- NII論文ID
- 110006436361
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- NII書誌ID
- AA10885497
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- ISSN
- 13475215
- 09186158
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- NDL書誌ID
- 8918936
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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