In Vivo AGO-APP For Cell-Type- and Compartment-Specific miRNA Profiling in the Mouse Brain
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AGO-APP through the expression of the T6B peptide permits the isolation of Ago-bound miRNAs. Here we present the generation and characterization of two transgenic mouse lines allowing to perform AGO-APP in vivo. First, we generated mice for CRE-dependent expression of T6B in the cytoplasm. Using this line we performed Ago-APP in olfactory bulb (OB) inhibitory interneurons and cerebral cortex excitatory neurons. Bioinformatic analysis validated the high reproducibility of the approach. It also demonstrated that, despite global miRNome conservation between the two cell types, a set of miRNAs including the miR-200 family and the miR-183/96/182 cluster, is massively enriched in OB interneurons which is consistent with previous observations. In the second mouse line T6B is fused to the postsynaptic protein PSD95. Isolation of T6B-PSD95 fractions from OB and cortical neurons identified specific sets of post-synapse enriched miRNAs. Gene ontology analyses confirmed that these miRNAs preferentially target mRNAs related to synaptic functions.
Motivation
Collectively, miRNAs are required for all biological processes in eukaryotes. However, due to a lack of appropriate methodological tools, the actual mode of action of individual miRNAs in a given cell is currently poorly understood. In this article, we describe a novel approach to study the specific expression of miRNAs in vivo, at the level of cell type and in a specific cell compartment.
