Lechler, Marion Brigitte (2021)
CRISPR/Cas9-mediated genome engineering of the SMARCB1 gene locus.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00011885
Dissertation, Erstveröffentlichung, Verlagsversion
Kurzbeschreibung (Abstract)
Truncating or nonsense mutations in the ubiquitously expressed tumour suppressor gene and BAF chromatin remodelling complex core subunit SMARCB1 cause malignant rhabdoid tumours (MRTs) of the brain, kidney, and the soft tissues. MRTs show a high clinical variance despite a simple cancer genome with SMARCB1 being the only gene recurrently mutated in these malignancies. MRTs of the brain are called atypical teratoid/rhabdoid tumours (AT/RT) and present a rare, but highly aggressive brain tumour entity with poor prognosis that predominantly occur in very young children. In this thesis, diverse constructs for the generation of three-colour-reporter cell lines with SMARCB1 mutations found in AT/RTs were established and methods required for the CRISPR/Cas9-mediated genome engineering using these constructs were optimised. Heterozygous missense mutations or small in-frame deletions clustering in the last two exons of the SMARCB1 gene are frequently observed in another disease entity, Coffin Siris-syndrome (CSS). CSS is a congenital neurodevelopmental disorder characterised by cognitive impairment, microcephaly, coarse facial features, and hypoplasia of the fifth finger- and/or toenail as well of the distal phalanx of the fifth finger and/or toe. CRISPR/Cas9 constructs for the generation of hiPSC and MCF10A cell lines with heterozygous c.1091_1093del AGA SMARCB1 mutation were generated to investigate the cellular consequences of this mutation. Comparison of cellular AT/RT and CSS models would contribute to a better understanding of how different types of SMARCB1 mutations can lead to two very different diseases and how particular mutations affect the gene expression of SMARCB1 itself. The recently published mouse model recapitulating SMARCB1-associated features of CSS, which is based on a partial loss-of-function of Smarcb1, revealed a diminished pool of neural progenitor cells (NPC) in the developing brain of these mice. To test the hypothesis if overexpression of Hes5 can rescue the impairment in mouse neural stem cells (NSC) maintenance, a lentiviral system for the overexpression of Cre recombinase and of Hes5 in Smarcb1(+/flox) NSCs and NPCs was optimised.
Typ des Eintrags: | Dissertation | ||||
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Erschienen: | 2021 | ||||
Autor(en): | Lechler, Marion Brigitte | ||||
Art des Eintrags: | Erstveröffentlichung | ||||
Titel: | CRISPR/Cas9-mediated genome engineering of the SMARCB1 gene locus | ||||
Sprache: | Englisch | ||||
Referenten: | Nuber, Prof. Dr. Ulrike A. ; Cardoso, Prof. Dr. M. Cristina | ||||
Publikationsjahr: | 2021 | ||||
Ort: | Darmstadt | ||||
Kollation: | X, 135 Seiten | ||||
Datum der mündlichen Prüfung: | 10 September 2020 | ||||
DOI: | 10.26083/tuprints-00011885 | ||||
URL / URN: | https://tuprints.ulb.tu-darmstadt.de/11885 | ||||
Kurzbeschreibung (Abstract): | Truncating or nonsense mutations in the ubiquitously expressed tumour suppressor gene and BAF chromatin remodelling complex core subunit SMARCB1 cause malignant rhabdoid tumours (MRTs) of the brain, kidney, and the soft tissues. MRTs show a high clinical variance despite a simple cancer genome with SMARCB1 being the only gene recurrently mutated in these malignancies. MRTs of the brain are called atypical teratoid/rhabdoid tumours (AT/RT) and present a rare, but highly aggressive brain tumour entity with poor prognosis that predominantly occur in very young children. In this thesis, diverse constructs for the generation of three-colour-reporter cell lines with SMARCB1 mutations found in AT/RTs were established and methods required for the CRISPR/Cas9-mediated genome engineering using these constructs were optimised. Heterozygous missense mutations or small in-frame deletions clustering in the last two exons of the SMARCB1 gene are frequently observed in another disease entity, Coffin Siris-syndrome (CSS). CSS is a congenital neurodevelopmental disorder characterised by cognitive impairment, microcephaly, coarse facial features, and hypoplasia of the fifth finger- and/or toenail as well of the distal phalanx of the fifth finger and/or toe. CRISPR/Cas9 constructs for the generation of hiPSC and MCF10A cell lines with heterozygous c.1091_1093del AGA SMARCB1 mutation were generated to investigate the cellular consequences of this mutation. Comparison of cellular AT/RT and CSS models would contribute to a better understanding of how different types of SMARCB1 mutations can lead to two very different diseases and how particular mutations affect the gene expression of SMARCB1 itself. The recently published mouse model recapitulating SMARCB1-associated features of CSS, which is based on a partial loss-of-function of Smarcb1, revealed a diminished pool of neural progenitor cells (NPC) in the developing brain of these mice. To test the hypothesis if overexpression of Hes5 can rescue the impairment in mouse neural stem cells (NSC) maintenance, a lentiviral system for the overexpression of Cre recombinase and of Hes5 in Smarcb1(+/flox) NSCs and NPCs was optimised. |
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Status: | Verlagsversion | ||||
URN: | urn:nbn:de:tuda-tuprints-118851 | ||||
Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie | ||||
Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Stammzell- und Entwicklungsbiologie |
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Hinterlegungsdatum: | 12 Feb 2021 07:46 | ||||
Letzte Änderung: | 16 Feb 2021 06:09 | ||||
PPN: | |||||
Referenten: | Nuber, Prof. Dr. Ulrike A. ; Cardoso, Prof. Dr. M. Cristina | ||||
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 10 September 2020 | ||||
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