Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.
Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.
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DOI：
10.1016/j.celrep.2017.03.048
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发表时间：
2017-04-18
期刊：
影响因子：
8.8
通讯作者：
Schmidt EF
中科院分区：
文献类型：
--
作者：
Nectow AR;Moya MV;Ekstrand MI;Mousa A;McGuire KL;Sferrazza CE;Field BC;Rabinowitz GS;Sawicka K;Liang Y;Friedman JM;Heintz N;Schmidt EF
翻译谱分析方法能够系统地表征复杂组织中的细胞类型，例如哺乳动物大脑，其中神经元分离是非常困难的。在这里，我们报告了一种通用的策略，以空间时间限制的方式，通过工程化的Cre依赖的腺相关病毒表达的EGFP标记的核糖体蛋白（AAV-FLEX-EGFPL 10a），以访问翻译mRNA的TRAP的CNS细胞类型。我们证明了这种AAV的效用，以靶向各种遗传和解剖学定义的表达Cre重组酶的神经群体，并说明了这种病毒TRAP（vTRAP）方法的能力，概括了通过bacTRAP在皮质丘脑神经元中跨多种血清型获得的分子谱。此外，空间限制AAV注射使得能够阐明该细胞类型内基因表达的区域差异。总之，这些结果确立了vTRAP策略对于可被工程化以表达Cre的任何CNS或外周细胞类型的分子解剖的广泛适用性。Nectow等人描述了vTRAP，一种以时空限制的方式从遗传上确定的细胞类型纯化翻译mRNA的技术。vTRAP与其他技术的多路复用提供了一种全面的策略来询问单个细胞类型特异性基因在神经回路功能中的确切作用。
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 to profile 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 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 AAV injections enabled the elucidation of regional differences in gene expression within this cell type. Taken together, 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. 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.