Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
12 2019
12 2019
Historique:
received:
04
04
2019
accepted:
03
09
2019
pubmed:
22
11
2019
medline:
8
2
2020
entrez:
22
11
2019
Statut:
ppublish
Résumé
The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.
Identifiants
pubmed: 31748752
doi: 10.1038/s41596-019-0241-8
pii: 10.1038/s41596-019-0241-8
pmc: PMC7089775
mid: NIHMS1555270
doi:
Substances chimiques
MicroRNAs
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
3538-3553Subventions
Organisme : NINDS NIH HHS
ID : K08 NS101091
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS116144
Pays : United States
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