High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.
Mol Cell
; 79(1): 167-179.e11, 2020 07 02.
Article
in En
| MEDLINE
| ID: mdl-32497496
ABSTRACT
The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombinant Fusion Proteins
/
RNA, Messenger
/
MicroRNAs
/
Embryonic Stem Cells
/
Argonaute Proteins
/
Glioma
Type of study:
Diagnostic_studies
Limits:
Animals
Language:
En
Journal:
Mol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2020
Document type:
Article
Affiliation country:
United States