@article{af7abdd3453b49c4a3fcb789b3f7b011,
title = "High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down",
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.",
keywords = "Ago2, CLIP, HaloTag, conditional, microRNAs, mouse, non-coding RNAs, targets",
author = "Xiaoyi Li and Yuri Pritykin and Concepcion, {Carla P.} and Yuheng Lu and {La Rocca}, Gaspare and Minsi Zhang and Bryan King and Cook, {Peter J.} and Au, {Yu Wah} and Olesja Popow and Paulo, {Joao A.} and Otis, {Hannah G.} and Chiara Mastroleo and Paul Ogrodowski and Ryan Schreiner and Haigis, {Kevin M.} and Doron Betel and Leslie, {Christina S.} and Andrea Ventura",
note = "Funding Information: We acknowledge the use of the Integrated Genomics Operation Core, which is funded by NCI Cancer Center Support Grant (CCSG) P30 CA08748 , Cycle for Survival , and the Marie-Jos{\'e}e and Henry R. Kravis Center for Molecular Oncology . This work was funded by grants from the NIH ( NCI R01CA149707 , A.V.), the Pershing Square Sohn Cancer Research Alliance (A.V.), the Starr Cancer Consortium (A.V. and D.B.), and the Geoffrey Beene Cancer Research Foundation (A.V.). Y.P. was supported by an AACR-Bristol-Myers Squibb immuno-oncology research fellowship (grant 19-40-15-PRIT ). C.P.C. was supported by NCI F31 training grant 1F31CA168356-01A1 . Funding Information: We acknowledge the use of the Integrated Genomics Operation Core, which is funded by NCI Cancer Center Support Grant (CCSG) P30 CA08748, Cycle for Survival, and the Marie-Jos?e and Henry R. Kravis Center for Molecular Oncology. This work was funded by grants from the NIH (NCI R01CA149707, A.V.), the Pershing Square Sohn Cancer Research Alliance (A.V.), the Starr Cancer Consortium (A.V. and D.B.), and the Geoffrey Beene Cancer Research Foundation (A.V.). Y.P. was supported by an AACR-Bristol-Myers Squibb immuno-oncology research fellowship (grant 19-40-15-PRIT). C.P.C. was supported by NCI F31 training grant 1F31CA168356-01A1. We thank Gregory Hannon for suggesting the use of the HaloTag to pull down Argonaute-containing complexes; Joana de Campos Vidigal for assistance with the design of the Halo-Ago2 targeting construct and gene targeting experiments; and members of the Ventura, Leslie, and Benezra laboratories for discussion and suggestions. Conceptualization, A.V. and C.S.L.; Methodology, X.L. C.P.C. Y.P. P.J.C. G.L.R. Y.L. K.M.H. D.B. A.V. and C.S.L.; Investigation, X.L, C.P.C. G.L.R. M.Z. B.K. P.J.C. Y.W.A. O.P. J.A.P. H.G.O. and R.S.; Formal Analysis and Software, Y.P. X.L. A.V. C.S.L. Y.L. and D.B.; Writing ? Original Draft, X.L. C.P.C. Y.P. C.S.L. and A.V.; Writing ? Review & Editing, X.L. C.P.C. Y.P. C.S.L. and A.V.; Funding Acquisition, A.V. C.S.L. D.B. and K.M.H.; Resources, P.O. and C.M.; Supervision, K.M.H. A.V. and C.S.L. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",
year = "2020",
month = jul,
day = "2",
doi = "10.1016/j.molcel.2020.05.009",
language = "English (US)",
volume = "79",
pages = "167--179.e11",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "1",
}