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Titre : | Obtention de mutants cellulaires et de vecteurs dexpression pour létude de mutants de ladénosine désaminase 1 |
Auteurs : | Anissa DADA, Auteur ; Thomas Michiels, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
ADAR1, adenosine deaminase acting on double-stranded RNA, is an enzyme that catalyses the reaction of deamination from adenosine to inosine. It mainly exists in 2 isoforms, the p110 and the p150. The shorter isoform, p110, is located in the nucleus, while the p150 is also found in the cytoplasm. The presence of double-stranded RNA in the cell triggers the innate immunity by MDA5 and PKR pathways. Even endogenous dsRNA is able to activate MDA5 and PKR, which could lead an autoimmune reaction. To avoid this, the enzyme ADAR1 converts adenosine to inosine in order to destabilize the dsRNA and prevent it from being recognized by MDA5 and PKR. Mutations in the ADAR1 gene cause the enzyme to lose its function, for example, this is the case in patients with the Aicardi-Goutières syndrome. This syndrome is an autoimmune disease characterized by the production of interferon leading to inflammation.
The aim of this work is divided into 3 points: - The first one is to generate ADAR1 KO cells by the CRISPR/Cas9 method and analyse them by Western Blot in order to observe the absence of the 2 isoforms. - The second one is to generate mutants of ADAR1 by PCR Overlap and then to clone the mutated fragments into pHS8 (which codes for p150) and pTC32 (which codes for p110) plasmids. The inserted mutations have been detected in patients with the Aicardi-Goutières syndrome, these are the substitution of a proline for alanine in position 193 and the substitution of an isoleucine for threonine in position 872. - The last one consists in the analysis of the expression and the localization of the mutants of ADAR1 after transfection of the mutants in the 293T ADAR1 KO cells. The CRISPR/Cas9 method made it possible to obtain 7 293T ADAR1 KO cell clones. The PCR Overlap + cloning led to the formation of 3 mutants: pHS8 with mutation Pro193Ala, pHS8 with mutation Ile872Thr and pTC32 with mutation Ile872Thr. The transfection of the mutants in the 293T ADAR1 KO cells shows that the mutations did not change the expression and localization of ADAR1. To conclude, the different stages of this work led to the production of 2 elements that will be used as a basis for subsequent analyzes in order to better understand the mechanism of action of the protein. Now that we are sure that the mutants of ADAR1 are expressed, it would be interesting to see if they have an effect on the cells during infection. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | Institut de Duve: Laboratoire de virologie |
Département : | Biologie médicale |
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TFE biologie médicale Adobe Acrobat PDF |