A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry

Scientific Reports
Seo Young KwakHyung Jun Ahn

Abstract

The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as ...Continue Reading

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Methods Mentioned

BETA
flow
imaging technique
in vitro transcription
PCR
xenograft
electrophoretic mobility shift assay
dynamic light scattering
Flow cytometry
ELISA
transfection

Software Mentioned

EZ
Image J
IVIS Spectrum
Capture
IVIS Living Imaging
Zetasizer Nano
Capture MG
ZEN
RNA mfold
mfold

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