A method for stabilizing RNA for transfection that allows control of expression duration

Authors

  • Toshinori Hayashi,

    1. Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington
    Current affiliation:
    1. Division of Biosignaling, School of Life Sciences, Tottori University Faculty of Medicine Yonago City, Tottori Prefecture, 683-8503 Japan.
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  • Deepak A. Lamba,

    1. Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington
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  • Amber Slowik,

    1. Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington
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    • Amber Slowik is a student in the graduate Program in Neurobiology and Behavior at the University of Washington.

  • Thomas A. Reh,

    1. Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington
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  • Olivia Bermingham-McDonogh

    Corresponding author
    1. Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington
    • Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington School of Medicine, 815 Mercer Street, Seattle, WA 98109
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Abstract

RNA transfection methods have not proven to be as popular as DNA methods due to the highly transient nature of the RNA inside the cell. However, there are many advantages in using RNA for gene over-expression, such as the rapidity of expression, the ability to express in all cell types without the need for cell-type-specific promoters, and the ability to analyze the effects of gene over-expression in a transient manner. Therefore, we have developed a method (StabiLizingUtr: SLU) to stabilize the RNA for varying durations, using specific sequences from the 3′UTR of the Venezuelan equine encephalitis virus (VEEV). We have designed a plasmid for cloning genes upstream from repeated stabilizing sequences to generate mRNA with one or more VEEV-stabilizing sequence motifs. We demonstrate this method in several cell and tissue types, including the mammalian cochlea, a tissue that has been difficult to transfect with other methods. Developmental Dynamics 239:2034–2010, 2010. © 2010 Wiley-Liss, Inc.

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