Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression
Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which...
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Published in | Nucleic acids research Vol. 43; no. 18; p. e120 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
15.10.2015
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Abstract | Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system. |
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AbstractList | Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system. |
Author | Yu, Han Jiang, Xiaoou Tan, Kar Tong Patzel, Volker Hang, Liting |
Author_xml | – sequence: 1 givenname: Han surname: Yu fullname: Yu, Han organization: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore – sequence: 2 givenname: Xiaoou surname: Jiang fullname: Jiang, Xiaoou organization: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore – sequence: 3 givenname: Kar Tong surname: Tan fullname: Tan, Kar Tong organization: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore – sequence: 4 givenname: Liting surname: Hang fullname: Hang, Liting organization: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore – sequence: 5 givenname: Volker surname: Patzel fullname: Patzel, Volker email: micvp@nus.edu.sg organization: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore micvp@nus.edu.sg |
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CitedBy_id | crossref_primary_10_1016_j_heliyon_2023_e16035 crossref_primary_10_1021_acsami_8b04294 crossref_primary_10_3390_pharmaceutics15020370 crossref_primary_10_1517_14712598_2016_1134480 crossref_primary_10_1016_j_omtm_2019_08_008 crossref_primary_10_1038_mt_2016_138 crossref_primary_10_1016_j_toxlet_2017_02_012 |
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SubjectTerms | Cell Line DNA - chemistry DNA Primers Furans - chemistry Gene Expression Gene Knockout Techniques Genetic Vectors - biosynthesis Genetic Vectors - chemistry Humans Methods Online Polymerase Chain Reaction - economics Polymerase Chain Reaction - methods RNA, Small Interfering - biosynthesis RNA, Small Interfering - genetics |
Title | Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression |
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