小鼠 TU 标签：一种用于纯化细胞类型特异性新生 RNA 的化学/遗传交叉方法。
Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.
机构信息
Institute of Neuroscience, University of Oregon, Eugene, 97403, USA.
出版信息
Genes Dev. 2013 Jan 1;27(1):98-115. doi: 10.1101/gad.205278.112.
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.
转录谱分析是一种理解发育和疾病的强大方法。目前的细胞类型特异性 RNA 纯化方法存在局限性，包括细胞解离创伤或无法识别所有 RNA 种类。在这里，我们描述了“小鼠硫代尿嘧啶（TU）标记”，这是一种用于体内标记和纯化细胞类型特异性 RNA 的遗传和化学交叉方法。Cre 诱导的尿嘧啶磷酸核糖基转移酶（UPRT）表达提供空间特异性；4-硫代尿嘧啶（4TU）的注射提供时间特异性。只有暴露于 4TU 的 UPRT(+)细胞会产生硫代 RNA，然后将其纯化用于 RNA 测序（RNA-seq）。这种方法可以从空间复杂和罕见（<5%）的细胞中纯化转录本，例如 Tie2:Cre(+)脑内皮细胞/小胶质细胞（76%通过表达模式验证），或者从急性诱导的转录本中纯化，例如脂多糖（LPS）注射。此外，通过 UPRT(+)骨髓移植生成嵌合小鼠可识别免疫细胞与龛位脾细胞的 RNA。TU 标记为在完整小鼠体内的特定时间特定细胞中鉴定活跃转录的基因提供了一种新方法。
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