Establishing an in vitro model for X chromosome reactivation in the germline
In female mammals, one of the X chromosomes is epigenetically inactivated through X chromosome Inactivation (XCI) during the epiblast differentiation. However, XCI is later specifically reversed in female germ cells by reactivation of the inactive X chromosome (XCR) via diffusible signaling molecule...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2019 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/667272 |
| Acceso en línea: | http://hdl.handle.net/10803/667272 |
| Access Level: | acceso abierto |
| Palabra clave: | Germ cells In vitro model Epigenetics X-chromosome X-chromosome reactivation Oligo-fish Células germinales Modelo in vitro Epigenética Reactivación del cromosoma X 575 |
| Sumario: | In female mammals, one of the X chromosomes is epigenetically inactivated through X chromosome Inactivation (XCI) during the epiblast differentiation. However, XCI is later specifically reversed in female germ cells by reactivation of the inactive X chromosome (XCR) via diffusible signaling molecules produced by female gonadal cells. The mechanistic study of XCR is however impeded by the low cell numbers present in vivo and the need for scalable readouts of X chromosome activity at allelic and single-cell resolution. We characterized XCI and XCR kinetics of a new cell differentiation protocol that differentiates embryonic stem cells (ESCs) into X-inactive epiblast-like cells (Epi-LCs), from which in vitro germ cell (PGC-LCs) differentiation is stimulated through a set of cytokines. The goal was to obtain a source of X-inactive PGC-LCs that could be characterized while exposed to XCR-inducing cues, conforming an In vitro model for germline XCR. Contarily to what was believed in the field, PGC-LCs could undergo XCR mediated by the cytokines present in the PGC-LC differentiation media without exposure to female gonadal cells. We also tested several oligo-FISH methodologies based on oligonucleotide labeling and hairpin chain reaction, discovering that combining hairpin chain reaction with the split-paired probe design of V3.0 smHCR provided a scalable platform to monitor the X activity at allelic resolution. This thesis allowed the implementation of an in vitro model for XCR characterization in the germline and a foundation for a scalable readouts of the X activity at allelic and single-cell resolution. |
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