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Title: Construction and characterization of synthetic microRNA cluster for multiplex RNA interference in mammalian cells      
dateReleased:
09-01-2016
description:
RNA interference (RNAi) technology is widely used in basic and translational research. By mimicking natural primary microRNA (pri-miRNA) cluster, multiple engineered hairpins can be transcribed as a single transcript from the same Pol-II promoter, enabling multiplex RNAi in mammalian cells 1-5. However, constructing synthetic miRNA cluster is still time-consuming, and the processing and function of miRNA cluster are incompletely understood. Here, we identified a miRNA precursor architecture that allowed precise miRNA maturation. We established a hierarchical cloning method for efficient construction of synthetic miRNA cluster harboring up to 18 miRNA precursors. We demonstrated that maturation and function of individual miRNA precursors were independent on their positions in the cluster. We then analyzed the integration efficiency of miRNA clusters with varying number of miRNA precursors by using CRISPR/Cas9 mediated integration, piggyBac transposon system, and lentiviral system. This synthetic miRNA cluster system provides an important tool for multiplex RNAi in mammalian cells. Different kinds of synthetic miRNAs or miRNA clusters, including the same synthetic miRNA precursor embedded in a series of derivative backbones, 18 synthetic miRNA precursors, miRNA precursors with various stem lengths and miRNA clusters carrying different numbers of different pri-miRNA hairpins, were expressed in HEK293 cells. The microRNA maturation pattern of individual miRNA precursors was detected by small RNA sequencing.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-71088
refinement:
raw
alternateIdentifiers:
71088
keywords:
functional genomics
dateModified:
09-12-2016
availability:
available
types:
gene expression
name:
Homo sapiens
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-71088/E-GEOD-71088.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-71088/E-GEOD-71088.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71088
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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