AGO-APP in vivo para el perfilado de ARNm específico de tipo celular y compartimento en el cerebro de ratón

AGO-APP through the expression of the T6B peptide permits the isolation of Ago-bound microRNAs (miRNAs). Here, we present the generation and characterization of two transgenic mouse lines that enable AGO-APP to be performed in vivo. First, we generated mice for CRE-dependent T6B expression throughout the cell. Using this line, we performed AGO affinity purification (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 aligns 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 postsynapse-enriched miRNAs. Gene ontology analyses confirmed that these miRNAs preferentially target mRNAs related to synaptic functions.