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- W2989657834 abstract "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. This protocol covers the preparation and use of artificial microRNA clusters for gene therapy. The in silico design of chimeric microRNA transgenes in which up to six natural microRNA hairpins are replaced by synthetic versions obviates the need for complex molecular cloning." @default.
- W2989657834 created "2019-12-05" @default.
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- W2989657834 date "2019-11-20" @default.
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- W2989657834 title "Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications" @default.
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- W2989657834 doi "https://doi.org/10.1038/s41596-019-0241-8" @default.
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