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

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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.