This issue is less of a concern for miRAP because Epigenetic inhibitor AGO2-miRNA interaction is very stable and of high affinity (Tang et al., 2008). In addition, comparison of FACS with miRAP in the Camk2α-Cre line suggests that

our method faithfully represent the miRNA profiles in this cell type. Discovered less than two decades ago, miRNAs have since been implicated in the regulation of almost all aspects of cellular processes. Despite their prominent expression in the mammalian brain, the role of miRNAs in brain development, function, and plasticity remains poorly understood. A major challenge is to link miRNA activity in defined neuron types to specific aspects of neuronal specification, development, and physiology; characterizing miRNA profiles in specific cell types is the first step. Using miRAP, we have obtained a set of miRNA expression profiles in defined neuron types in mouse brain. Our study reveals the expression of a large fraction of known miRNAs with distinct profiles in glutamatergic and GABAergic neurons and subtypes of GABAergic neurons. We have further PLX4032 in vitro detected putative novel miRNAs, tissue- or cell-type-specific strand selection of miRNAs, and miRNA editing. This generally applicable miRAP method will facilitate a systematic

analysis of miRNA expression and regulation in specific neuron types in the context of neuronal specification, development, physiology, plasticity, pathology, and disease models. Targeting of rare cell types may further reveal novel, low abundant miRNAs and link novel regulatory mechanisms

such as miRNA editing to specific neuronal and circuitry function. Identification of mRNA targets in defined cell types is key to understanding the biological function of miRNAs. As miRNA activity requires base-pairing with only 6–8 nucleotides of mRNA, target prediction through bioinformatics has proven to be challenging. Recently, Ago HITS-CLIP has been used to profile miRNA-mRNA targets in the mouse brain (Chi et al., 2009). Genetically targeted miRAP provides a possibility for cell-type-specific Ago2 HITS-CLIP using the MYC or GFP antibody. To generate mouse line that conditionally express GFP –myc-Ago2, a cassette containing LoxP-STOP -LoxP-GFP-myc-Ago2 was cloned in to a Rosa26-CAG targeting vector which contains DTA negative selection marker. GFP-myc-Ago2 is Montelukast Sodium a gift from Dr. Richard M. Schultz in University of Pennsylvania. The STOP cassette contains Neo gene which confers G418 resistance. The targeting vector was linearized by PacI digestion and transfected into C57BL/6 mouse ES cell line. G418-resistant ES clones were screened by PCR first using a forward primer upstream of 5′ homologous arm and a reverse primer in the transgene promoter region, then confirmed by Southern blot of EcoRV digested DNA, which was probed by a 134 bp genomic fragment upstream of the 5′ targeting arm. All PCR positive clones were also positive for Southern blot.