Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP

• Published in: Cell reports (Cambridge) 2017-04, Vol.19 (3), p.655-667
• Main Authors: Nectow, Alexander R., Moya, Maria V., Ekstrand, Mats I., Mousa, Awni, McGuire, Kelly L., Sferrazza, Caroline E., Field, Bianca C., Rabinowitz, Gabrielle S., Sawicka, Kirsty, Liang, Yupu, Friedman, Jeffrey M., Heintz, Nathaniel, Schmidt, Eric F.
• Format: Article
• Language: English
• Subjects: AAV; Animals; Biomarkers - metabolism; Chromatography, Affinity - methods; corticothalamic; Dependovirus - metabolism; Female; Gene Expression Regulation; Green Fluorescent Proteins - metabolism; Hypothalamic Hormones - metabolism; Hypothalamus - metabolism; Male; MCH; Melanins - metabolism; Mice; molecular profiling; Neurons - metabolism; Ntsr1; Pituitary Hormones - metabolism; Protein Biosynthesis; Reproducibility of Results; Ribosomes - metabolism; RNA-seq; Serotyping; TRAP; Viruses - metabolism
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2017.03.048

Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. [Display omitted] •vTRAP enables rapid, viral-based tagging of translating ribosomes in defined cells•vTRAP faithfully recapitulates molecular profiles obtained from bacTRAP•vTRAP can detect regional, cell-type-specific differences in gene expression•vTRAP was used for comprehensive molecular profiling of LH MCH neurons Nectow et al. describe vTRAP, a technology to purify translating mRNAs from genetically defined cell types in a spatiotemporally restricted fashion. Multiplexing vTRAP with other technologies offers a comprehensive strategy to interrogate the precise role of individual, cell-type-specific genes in neural circuit function.