Bioluminescence as a tool for studying imprinted gene expression

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.Open Acces

Outcomes following kidney transplantation in patients with sickle cell disease:The impact of automated exchange blood transfusion

There are over 12,000 people with sickle cell disease (SCD) in the UK, and 4-12% of patients who develop Sickle Cell Nephropathy (SCN) progress to End Stage Renal Disease (ESRD). Renal transplantation offers the best outcomes for these patients with but their access to transplantation is often limited. Regular automated exchange blood transfusions (EBT) reduce the complications of SCD and may improve outcomes. However, concerns over alloimmunisation limit its widespread implementation. In this retrospective multicenter study, data were collected on 34 SCD patients who received a kidney transplant across 6 London Hospitals between 1997 and 2017. 20/34 patients were on an EBT program, pre or post renal transplantation. Overall patient and graft survival were inferior to contemporaneous UK data in the ESRD population as a whole, a finding which is well-recognised. However, patient survival (CI 95%, p = 0.0032), graft survival and graft function were superior at all time-points in those who received EBT versus those who did not. 4/20 patients (20%) on EBT developed de novo donor specific antibodies (DSAs). 3/14 patients (21%) not on EBT developed de novo DSAs. The incidence of rejection in those on EBT was 5/18 (28%), as compared with 7/13 (54%) not on EBT. In conclusion, our data, while limited by an inevitably small sample size and differences in the date of transplantation, do suggest that long-term automated EBT post renal transplant is effective and safe, with improvement in graft and patient outcomes and no increase in antibody formation or graft rejection