Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors
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 deliv...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2019-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050119300889 |
| _version_ | 1811286663359365120 |
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| author | Samantha Leeanne Cyrill Avantika Ghosh Pei She Loh Genim Siu Xian Tan Volker Patzel |
| author_facet | Samantha Leeanne Cyrill Avantika Ghosh Pei She Loh Genim Siu Xian Tan Volker Patzel |
| author_sort | Samantha Leeanne Cyrill |
| collection | DOAJ |
| description | 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. Keywords: dumbbell vector, DNA minimal vector, cloning-free generation, small RNA expression, high-throughput-compatible vector production, functional genomics, gene knockdown in primary cells, small hairpin RNA, microRNA |
| first_indexed | 2024-04-13T03:03:57Z |
| format | Article |
| id | doaj.art-2c70c30120dd41b3a628ab6200763a50 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-04-13T03:03:57Z |
| publishDate | 2019-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-2c70c30120dd41b3a628ab6200763a502022-12-22T03:05:19ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012019-12-0115149156Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA VectorsSamantha Leeanne Cyrill0Avantika Ghosh1Pei She Loh2Genim Siu Xian Tan3Volker Patzel4Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, SingaporeDepartment of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, SingaporeDepartment of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, SingaporeDepartment of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, SingaporeDepartment of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, Singapore; Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK; Corresponding author: Volker Patzel, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117545, Singapore.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. Keywords: dumbbell vector, DNA minimal vector, cloning-free generation, small RNA expression, high-throughput-compatible vector production, functional genomics, gene knockdown in primary cells, small hairpin RNA, microRNAhttp://www.sciencedirect.com/science/article/pii/S2329050119300889 |
| spellingShingle | Samantha Leeanne Cyrill Avantika Ghosh Pei She Loh Genim Siu Xian Tan Volker Patzel Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors Molecular Therapy: Methods & Clinical Development |
| title | Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors |
| title_full | Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors |
| title_fullStr | Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors |
| title_full_unstemmed | Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors |
| title_short | Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors |
| title_sort | universal template assisted cloning free method for the generation of small rna expressing dumbbell shaped dna vectors |
| url | http://www.sciencedirect.com/science/article/pii/S2329050119300889 |
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