In utero electroporation-based translating ribosome affinity purification identifies age-dependent mRNA expression in cortical pyramidal neurons

Publication date: Available online 29 May 2018
Source:Neuroscience Research
Author(s): Tianxiang Huang, Lena Nguyen, Tiffany V. Lin, Xuan Gong, Longbo Zhang, Gi Bum Kim, Matthew R. Sarkisian, Joshua Breunig, Angelique Bordey
We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages—P0, P7, and P14—when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ∼7,300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.



https://ift.tt/2JqmtVP