Publicação
Intersectional genetics system for stem cell selection
| datacite.subject.fos | Departamento de Biologia Vegetal | pt_PT |
| dc.contributor.advisor | Rodrigues, Maria Gabriela, 1965- | |
| dc.contributor.advisor | Gontijo, Alisson Marques de Miranda Cabral | |
| dc.contributor.author | Nabo, João Manuel Cavaco Cota Paussão | |
| dc.date.accessioned | 2024-02-14T15:58:05Z | |
| dc.date.available | 2024-02-14T15:58:05Z | |
| dc.date.issued | 2023 | |
| dc.date.submitted | 2023 | |
| dc.description | Tese de mestrado, Biologia Molecular e Genética, 2023, Universidade de Lisboa, Faculdade de Ciências | pt_PT |
| dc.description.abstract | Stem cell therapy holds promise for treating conditions like macular degeneration, stroke, and diabetes. In most of these therapies, replenishing a target cell type is desired. In contrast, the undifferentiated cells or differentiated cells of the wrong lineage are unnecessary or can lead to side effects such as teratoma formation. One way to overcome this is to engineer the transplanted cells to carry a functional genetic cell ablation system in all transplanted cells except for the target cell type. Versatile Entry Codes (VEnCodes) based on the intersection of regulatory elements can enable selective gene delivery to specific cell types. This study explores two alternative approaches to eliminate undesired cells while safeguarding cells of interest selectively: an inducible Caspase 9 (iCasp9)-mediated cell death system and a VEnCode-compatible split-Cre recombinase (Split-Cre-VEnCode) system. The iCasp9 system used the TetOn system with rtTA as the input and iCasp9 as the output. By precisely controlling iCasp9 expression levels and activity using doxycycline and the chemical inducer of dimerization AP20187, respectively, the system successfully induced cell death in HEK-293T cells. Whereas further testing of the drug AP20187 dosage is required to increase cell death, these promising results warrant future investigations in combination with the VEnCode system and different cell types to assess this system's potential applications fully. The Split-Cre-VEnCode system utilized LoxP sites strategically placed to manipulate downstream gene expression. Successful DNA recombination events were observed by monitoring the expression of mScarlet-I as a reporter gene. The system demonstrated inversion and deletion capabilities, offering controlled on/off effects. However, optimization is still required to split the Cre recombinase into >2 non-complementing fragments to unlock the full capabilities of this Split-Cre-VEnCode system. While this study primarily evaluated the systems in HEK-293T cells, future studies should validate their functionality in diverse cell lines. Additionally, long-term effects, stability, kinetics of activation, and potential off-target effects need to be investigated. These findings highlight the potential of these ablation systems complemented with VEnCode-based biosensor prototypes to enhance the specificity and safety of genetic delivery systems, providing powerful tools for conditional gene therapy and precise cellular ablation in biomedical research. iCasp9-mediated cell death and split-Cre recombinase systems are potential tools for cell ablation and targeted gene expression. | pt_PT |
| dc.identifier.tid | 203524187 | |
| dc.identifier.uri | http://hdl.handle.net/10451/62624 | |
| dc.language.iso | eng | pt_PT |
| dc.subject | Caspase 9 indutível | pt_PT |
| dc.subject | Split-Cre | pt_PT |
| dc.subject | morte celular | pt_PT |
| dc.subject | terapias celulares | pt_PT |
| dc.subject | sistema VEnCode | pt_PT |
| dc.subject | Teses de mestrado - 2023 | pt_PT |
| dc.title | Intersectional genetics system for stem cell selection | pt_PT |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | openAccess | pt_PT |
| rcaap.type | masterThesis | pt_PT |
| thesis.degree.name | Tese de mestrado em Biologia Molecular e Genética | pt_PT |