A mouse model of RB1 imprinting: knock-in of human PPP1R26P1 into mouse Rb1

RB1印记小鼠模型：将人PPP1R26P1敲入小鼠Rb1

• 批准号: 251565676
• 负责人: Professorin Dr. Laura Steenpaß
• 金额: --
• 依托单位: Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251565676?language=en
• 关键词: mouse; model; RB1; imprinting; knock

The human retinoblastoma gene (RB1) is imprinted; the mouse Rb1 gene is not. Imprinted expression of RB1 is due to differential methylation of a CpG island (CpG85), which is located in the pseudogene PPP1R26P1 in intron 2 of RB1. We used homologous recombination to introduce human PPP1R26P1 into intron 2 of the mouse Rb1 in embryonic stem cells. We showed that transcription from the unmethylated CpG85 in PPP1R26P1 takes place and observed reduced expression of full-length Rb1 from the targeted allele. Our results identify human PPP1R26P1 as a cis-repressive element in murine ES cells. Using the modified ES cells, we generated mice carrying the human PPP1R26P1 in intron 2 of Rb1. The aim of this proposal is to test if transmission of the human PPP1R26P1 through the mouse germline results in imprinting of Rb1. We will determine the acquisition of DNA methylation in the germline of both sexes, and we will analyze whether CpG85 is subjected to differential DNA methylation as it is in humans. Following this, we will determine whether the alternative Rb1 transcript is expressed and if this results in Rb1 repression in cis. The experiments outlined here will lead to a deeper understanding of genomic imprinting by the integration of retroelements and will provide us with a model to study parent-of-origin effects in retinoblastoma.

人类视网膜母细胞瘤基因（RB 1）是印记的;小鼠Rb 1基因不是。RB 1的印记表达是由于CpG岛（CpG 85）的差异甲基化，其位于RB 1内含子2的假基因PPP 1 R26 P1中。我们利用同源重组技术将人PPP 1 R26 P1基因导入小鼠胚胎干细胞Rb 1基因的内含子2中。我们发现PPP 1 R26 P1中非甲基化CpG 85的转录发生，并观察到靶向等位基因的全长Rb 1表达减少。我们的研究结果确定人类PPP 1 R26 P1作为一个顺式抑制元件在小鼠ES细胞。使用修饰的ES细胞，我们产生了在Rb 1的内含子2中携带人PPP 1 R26 P1的小鼠。本提案的目的是测试人PPP 1 R26 P1通过小鼠生殖系的传播是否会导致Rb 1的印迹。我们将确定两性生殖系中DNA甲基化的获得，并分析CpG 85是否像人类一样受到差异DNA甲基化的影响。在此之后，我们将确定是否表达替代Rb 1转录本，以及这是否导致Rb 1顺式阻遏。这里概述的实验将导致更深入地了解基因组印迹的整合retroelements，并将为我们提供一个模型来研究父母的起源在视网膜母细胞瘤的影响。