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

人视网膜母细胞瘤基因（RB1）被印迹；而小鼠的Rb1基因则不是。RB1的印迹表达是由于位于RB1内含子2的假基因PPP1R26P1上的CpG岛（CpG85）的差异甲基化。我们利用同源重组将人PPP1R26P1引入小鼠胚胎干细胞Rb1的内含子2中。我们发现PPP1R26P1中未甲基化的CpG85发生了转录，并且观察到来自目标等位基因的全长Rb1的表达减少。我们的研究结果确定人类PPP1R26P1是小鼠ES细胞中的顺式抑制元件。利用修饰的胚胎干细胞，我们在Rb1的内含子2中产生了携带人类PPP1R26P1的小鼠。这项提议的目的是测试人类PPP1R26P1通过小鼠种系传播是否会导致Rb1的印记。我们将确定两性种系中DNA甲基化的获得，我们将分析CpG85是否受到与人类不同的DNA甲基化。接下来，我们将确定替代Rb1转录本是否表达，以及这是否导致顺式Rb1抑制。这里概述的实验将通过整合逆转录因子导致对基因组印迹的更深入理解，并将为我们研究视网膜母细胞瘤的亲本起源效应提供一个模型。

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.