Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.omtm.2019.08.008
Title: Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors
Authors: Cyrill, S.L.
Ghosh, A. 
Loh, P.S. 
Tan, G.S.X.
Patzel, V. 
Keywords: cloning-free generation
DNA minimal vector
dumbbell vector
functional genomics
gene knockdown in primary cells
high-throughput-compatible vector production
microRNA
small hairpin RNA
small RNA expression
Issue Date: 2019
Publisher: Cell Press
Citation: Cyrill, S.L., Ghosh, A., Loh, P.S., Tan, G.S.X., Patzel, V. (2019). Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors. Molecular Therapy - Methods and Clinical Development 15 : 149-156. ScholarBank@NUS Repository. https://doi.org/10.1016/j.omtm.2019.08.008
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
Abstract: Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells. © 2019 The Authors
Source Title: Molecular Therapy - Methods and Clinical Development
URI: https://scholarbank.nus.edu.sg/handle/10635/209879
ISSN: 2329-0501
DOI: 10.1016/j.omtm.2019.08.008
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
Appears in Collections:Elements
Staff Publications

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
10_1016_j_omtm_2019_08_008.pdf1.51 MBAdobe PDF

OPEN

NoneView/Download

SCOPUSTM   
Citations

1
checked on May 14, 2022

Page view(s)

28
checked on May 12, 2022

Google ScholarTM

Check

Altmetric


This item is licensed under a Creative Commons License Creative Commons