Institute of Neuroscience, University of Oregon, Eugene, OR 97403.
Department of Biology, University of Oregon, Eugene, OR 97403.
eNeuro. 2017 Oct 4;4(5). doi: 10.1523/ENEURO.0181-17.2017. eCollection 2017 Sep-Oct.
Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, , to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the transgenic mouse to a line to express UPRT in layer 2/3 of visual cortex or to an line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.
硫代尿嘧啶（TU）标记是一种在特定细胞类型中共价标记新转录 RNA 的交叉方法。细胞类型特异性是通过靶向表达酶尿嘧啶磷酸核糖基转移酶（UPRT）产生的；时间特异性是通过修饰的尿嘧啶类似物 4TU 的脉冲产生的。该技术已在小鼠中使用 Cre 依赖性 UPRT 转基因 ，用于分析内皮细胞中的 RNA，但仍未测试 4TU 是否可以穿过血脑屏障（BBB），或者该转基因是否可用于纯化神经元 RNA。在这里，我们将 转基因小鼠与 线杂交，在视觉皮层的第 2/3 层表达 UPRT，或与 线杂交，在视觉皮层的第 4 层表达 UPRT。我们从出生后第 12 天（P）的两种基因型以及不表达 UPRT 的野生型（WT）小鼠（背景对照）中纯化硫代标记的 mRNA。我们发现，将 Sepw1 纯化的 RNA 与 WT 或 Nr5a1 纯化的 RNA 进行比较，使我们能够鉴定出富含视觉皮层第 2/3 层的基因。在这里，我们表明 Cre 依赖性 UPRT 表达可用于从完整的小鼠大脑中纯化细胞类型特异性 mRNA，并提供了 4TU 可以穿过 BBB 标记体内 RNA 的第一个证据。
