transgenic expression of UPRT as a novel tool for tagging RNA in specific tissues of the mouse

UPRT 的转基因表达作为在小鼠特定组织中标记 RNA 的新工具

• 批准号: BB/K013424/1
• 负责人: Martin Turner
• 金额: $ 13.98万
• 依托单位: Babraham Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK013424%2F1
• 关键词: transgenic; expression; UPRT; novel; tool

Summary (Lay)RNA is synthesised using information encoded by DNA and it mediates many of the functions of the genome as well as producing proteins. Understanding the regulation of RNA is a priority for the understanding of cell function. Like DNA sequences, RNA sequences are made up for four bases, but, unlike DNA, RNA contains the nucleoside uridine which is composed of the pyrimidine uracil. Uracil is not incorporated into the RNA of mouse or human cells as the required enzymes for this pathway are not encoded in the mammalian genome. Consequently, controlled expression of the enzyme uracil phosphoribosyltransferase (UPRT) which converts uracil into uridine monophosphate allows the incorporation of uracil in cell-type specific or temporally controlled manner labelling of RNA. We have generated a transgenic mouse that expresses UPRT in a controllable way and we want to demonstrate the broad utility of this as a tool for purification of RNA or the identification of RNA protein interactions in rare cell subsets in complex mixtures of cells. This is an improvement because it will open access to the RNA makeup of some cell types that cannot be easily purified. Furthermore, the act of purification is known to change the quantity and quality of the RNA within the cell. Thus by reducing the numbers of manipulations a more accurate readout of RNA content or RNA-protein interactions will be obtained.

RNA是利用DNA编码的信息合成的，它介导基因组的许多功能以及产生蛋白质。理解RNA的调节是理解细胞功能的优先事项。与DNA序列一样，RNA序列由四个碱基组成，但与DNA不同的是，RNA含有由嘧啶尿嘧啶组成的核苷尿苷。尿嘧啶不掺入小鼠或人类细胞的RNA中，因为该途径所需的酶不在哺乳动物基因组中编码。因此，将尿嘧啶转化为尿苷单磷酸的酶尿嘧啶磷酸核糖基转移酶（UPRT）的受控表达允许以细胞类型特异性或时间受控的方式标记RNA掺入尿嘧啶。我们已经产生了一个转基因小鼠，表达UPRT在一个可控的方式，我们想证明这作为一种工具，用于纯化RNA或RNA蛋白相互作用的鉴定在罕见的细胞亚群中的复杂的细胞混合物的广泛用途。这是一种改进，因为它将开放对一些不能容易纯化的细胞类型的RNA组成的访问。此外，已知纯化行为会改变细胞内RNA的数量和质量。因此，通过减少操作次数，将获得更准确的RNA含量或RNA-蛋白质相互作用读数。