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Induction of site-specific methylation in induced pluripotent stem cells of a patient with Angelman syndrome
In this study, an iPSC model of Angelman syndrome was developed. iPSCs were generated from an Angelman syndrome patient harbouring a three-base pair deletion in the maternal UBE3A allele and from a healthy control person. The maternal UBE3A allele is expressed but the protein UBE3A is not functional. Therefore, the cells exhibit normal expression of all genes at the PWS/AS locus. In comparison with all iPSC lines generated as a model of Angelman syndrome so far, this is a significant advantage because all observed effects may be attributed directly and solely to the inactive UBE3A protein. Using lentiviral transduction, we reprogrammed fibroblasts from the patient and from the healthy control person, and isolated several clones. Their thorough characterisation followed confirming their pluripotency, identity and integrity by various methods. Based on the results, clones most suitable for further analyses were selected. The stability of the imprint at the PWS/AS locus is a critical prerequisite for the faithful analysis of imprinted expression and for the induction of DNA methylation on the paternal allele. Therefore, the methylation status at six imprinted loci and two pluripotent loci was determined in the iPSCs generated at the beginning of this project, their parental fibroblasts, in hESCs H1 and H9, and in published cell lines AGI-0 and MCH2-10 that represented iPSC controls. Most importantly, I observed an exceptional stability of the methylation imprint at PWS-SRO. Three imprinted loci showed a tendency towards a loss of methylation and two exhibited susceptibility to acquiring methylation. Both pluripotent loci displayed a higher level of methylation in differentiated cells than in pluripotent cells. My results on the stability of the assayed loci upon reprogramming are in agreement with previously reported observations. In cooperation with Anika Neureiter from the Institute for Transfusion Medicine, iPSCs were differentiated into neurons. We determined by single-nucleotide primer extension assay that ...
Induction of site-specific methylation in induced pluripotent stem cells of a patient with Angelman syndrome
In this study, an iPSC model of Angelman syndrome was developed. iPSCs were generated from an Angelman syndrome patient harbouring a three-base pair deletion in the maternal UBE3A allele and from a healthy control person. The maternal UBE3A allele is expressed but the protein UBE3A is not functional. Therefore, the cells exhibit normal expression of all genes at the PWS/AS locus. In comparison with all iPSC lines generated as a model of Angelman syndrome so far, this is a significant advantage because all observed effects may be attributed directly and solely to the inactive UBE3A protein. Using lentiviral transduction, we reprogrammed fibroblasts from the patient and from the healthy control person, and isolated several clones. Their thorough characterisation followed confirming their pluripotency, identity and integrity by various methods. Based on the results, clones most suitable for further analyses were selected. The stability of the imprint at the PWS/AS locus is a critical prerequisite for the faithful analysis of imprinted expression and for the induction of DNA methylation on the paternal allele. Therefore, the methylation status at six imprinted loci and two pluripotent loci was determined in the iPSCs generated at the beginning of this project, their parental fibroblasts, in hESCs H1 and H9, and in published cell lines AGI-0 and MCH2-10 that represented iPSC controls. Most importantly, I observed an exceptional stability of the methylation imprint at PWS-SRO. Three imprinted loci showed a tendency towards a loss of methylation and two exhibited susceptibility to acquiring methylation. Both pluripotent loci displayed a higher level of methylation in differentiated cells than in pluripotent cells. My results on the stability of the assayed loci upon reprogramming are in agreement with previously reported observations. In cooperation with Anika Neureiter from the Institute for Transfusion Medicine, iPSCs were differentiated into neurons. We determined by single-nucleotide primer extension assay that ...
Induction of site-specific methylation in induced pluripotent stem cells of a patient with Angelman syndrome
Stanurova, Jana (Autor:in) / Horsthemke, Bernhard
07.11.2017
Hochschulschrift
Elektronische Ressource
Englisch
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