Bioluminescence as a tool for studying imprinted gene expression

Abstract

Genomic imprinting is an epigenetic phenomenon which orchestrates allele specific expression of a subset of genes according to parental origin. Monoallelic expression is directed by imprinting control centres and is established in the germ line through acquisition of differential DNA methylation. Even though they represent a small subset of genes within the mammalian genome, imprinted genes have critical roles in an array of important biological functions including metabolism, growth and behaviour. Even though the evolutionary rationale for the functional haploidy of these gene is uncertain, the consequences of dysregulation of some imprinted genes demonstrate that gene dosage is important. Cdkn1c is a maternally expressed gene which has an important role in foetal development, behaviour and metabolism and has been shown to be sensitive to gene dosage alteration. In this thesis, I first demonstrate that embryonic germ cells, on fusion with somatic mouse B cells can, not only dominantly reprogram them, but can induce imprint erasure at the gametic DMR governing imprinted expression of Cdkn1c. I also describe the development and characterisation of a mouse line in which firefly luciferase was knocked in to endogenous mouse Cdkn1c. I demonstrate that bioluminescence imaging can be used to provide a non-invasive read out of endogenous imprinted gene expression thus providing an opportunity to not only monitor expression longitudinally but also provide an opportunity to study the consequences of imprint perturbation in vivo. Furthermore, the reporter was expressed in a parent-of-origin manner and heterozygotes were found to have wild type levels of transcript and DNA methylation at DMRs. Using this model, transient de-repression of the paternal allele by chromatin modifying drugs could by visualised non-invasively via bioluminescence in vivo in embryos. This work demonstrates that these reporter mouse models can be used to interrogate the effects of in utero environmental stressors on imprinted gene expression.