Assisted reproduction in humans has been linked to loss-of-imprinting in gametes and/or preimplantation embryos that results in adverse consequences during fetal and postnatal development, the most prevalent being Beckwith-Wiedemann Syndrome (BWS). The immediate goal of this proposal is to establish a loss-of- imprinting model that recapitulates BWS. The proposed model will enable the long-term goal of elucidating the mechanisms by which assisted reproductive technologies can induce loss-of-imprinting syndromes, and most importantly, induce the resulting phenotypes. BWS is an overgrowth condition with an increased incidence of neonatal neoplasia. This syndrome has been recognized as being the result of misregulation of imprinted gene expression. Imprinting is defined as allelic-specific gene expression. Imprinted genes are frequently found in clusters and many are involved in regulating growth and development of the fetus and placenta. The clusters'regulatory regions, known as imprinting control regions (ICR), are marked in a parent-specific manner, usually by DNA methylation. These marks orchestrate allelic expression of each gene within the cluster. The inability to maintain the imprinting mark during development and the resulting misregulation of the cluster's genes have been associated with several human syndromes, one of which is BWS. Similar to humans, assisted reproduction in bovids can cause an overgrowth syndrome phenotypically similar to BWS and historically referred to as Large Offspring Syndrome (LOS). This proposal intends to develop a bovine model to investigate the relationship between loss-of-imprinting and the overgrowth phenotype.
Specific Aim 1 will develop assessments for evaluating allele-specific imprinted gene status in this model. This will be accomplished by characterization of distinguishing DNA polymorphisms (by PCR and sequencing) in BWS- associated genes in Bos taurus and Bos indicus. The identified polymorphisms will be used to develop protocols to distinguish the parental alleles of B. taurus X B. indicus F1 individuals.
Specific Aim 2 will determine ICR methylation and allelic expression of BWS-associated genes in F1 individuals produced through natural conception as a baseline of """"""""normal."""""""" Parent-specific ICR methylation will be independently analyzed by restriction digest and sequencing of bisulfite converted, PCR amplified DNA in placental and fetal tissues of F1 concepti.
Specific Aim 3 will validate the model by determining if loss-of-imprinting of these genes is associated with the overgrowth phenotype. This will be accomplished by generating bovine embryos in vitro under conditions known to induce LOS. The resulting embryos will be transferred and allowed develop to day 100 of gestation. Placental and fetal tissues will be analyzed at BWS-associated imprinted loci and compared to """"""""normal"""""""". Correlations will be assessed between loss-of-imprinting and phenotype. Preliminary data demonstrates a very good likelihood of success for this model.
Assisted reproduction in humans has been linked to loss-of-imprinting in gametes and/or preimplantation embryos that results in adverse consequences during fetal and postnatal development, the most prevalent being Beckwith-Wiedemann Syndrome (BWS). BWS is characterized by fetal and neonatal overgrowth and an increased likelihood of childhood tumors. Since this phenotype is only observed in ruminants and humans as a result of assisted reproduction, we propose to develop a bovine model to investigate the relationship between loss-of-imprinting and the overgrowth phenotype. The proposed model will enable the long-term goal of elucidating the mechanisms by which assisted reproductive technologies can induce loss-of-imprinting syndromes, and most importantly, induce the resulting phenotypes.
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