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Identifier 000402145
Title Employing CRISPR/Cas9 to study the GLUD1 and /or GLUD2 knock out effect on HEK293 cell cultures
Alternative Title Χρήση του CRISPR/Cas9 για τη μελέτη της επίδρασης που προκαλεί η απαλοιφή του GLUD1 ή/και του GLUD2 γονιδίου σε HEK293 κυτταροκαλλιέργειες
Author Κοσμοπούλου Χριστίνα
Thesis advisor Ζαγανάς, Ιωάννης
Reviewer Καραγωγέως, Δόμνα
καρδάσης, Δημήτριος
Abstract Background: Glutamate dehydrogenase (GDH) is an abundant mitochondrial enzyme that catalyzes the reversible interconversion of glutamate into a-ketoglutarate and ammonia, using NADP(H) and NAD(H) as cofactors. In mammals, GDH is believed to favor the production of a-ketoglutarate which in turn is used for energy production through its entry to the Krebs cycle. In humans, GDH is encoded by two different genes, GLUD1 and GLUD2, that are highly homologous. Aim of the study: Aim of this study was to examine the effect on cell viability and metabolism of knocking out either GLUD1 or GLUD2 (or both) in human cell cultures. Methods: In the current study we employed the CRISPR/Cas9 system (a new highly efficient system of gene elimination that is increasingly used in molecular biology) to delete either both or each one of theGLUD1 and GLUD2genes at a time, in HEK293 cell cultures. For this reason, two pairs of complementary single guide RNAs (sgRNAs) specific for hGDH1 and two pairs specific for hGDH2 were designed. Then, each sgRNA was cloned into pX458 plasmid vector that contains the Cas9 gene. Results and discussion: Due to limited time, we did not reach the stage of expressing these plasmids in HEK293 cells, (this is currently pursued).The final aim is to study cell viability and metabolic profile, after eliminating hGDHs from these HEK293 cells.
Language English
Subject Γλουταμική αφυδρογονάση
Issue date 2016-07-19
Collection   Faculty/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
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