Akademik Veri Yönetim Sistemi
Tez Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: İngiltere
Tez Danışmanı: Khuloud Al-Jamal
Tezin Onay Tarihi: 2017
Tezin Dili: İngilizce
Desteklendiği Program: Diğer
Özet:
CRISPR can be used to target any gene by using a DNA targeting short guide RNA sequence (gRNA), which is associated with a DNA nuclease (Cas9). It is therefore a promising approach for many disease-related studies, especially cancer therapy. To deliver Cas9 to its target, a biocompatible and stable delivery system is needed. In this regard, functionalized carbon nanotubes (f-CNTs) are promising carriers since they can pass through membranes and reach the brain. In this study, three different gRNA sequences targeting firefly luciferase (a reporter gene) were designed, aiming at using it for quick in vitro and in vivo assessment of gene targeting. The transfection protocol (which uses commercially available Lipofectamine RNAiMAX® ) was validated in GL261-luc glioma cells using a validated gRNA (HPRT), which was followed by investigation of luciferase gRNA cutting efficiencies (by PCR and agarose gel elctrophoresis). The 'reverse' transfection protocol in media without serum (for 4h) was found to be the most efficient method for Cas9-mediated editing in GL261-luc. Moreover, no toxicity was detected in cells transfected by this method. However, no indel formation (gene cutting) was detected with the designed luciferase gRNAs. Luciferase assay was performed for expression analysis and the results demonstrated that protein expression was not disrupted. Simultaneously, CNTs of two different diameters were functionalized via oxidation, amidation, or amine-functionalization and further conjugated to Cas9. Efficient CNT functionalization was achieved and results demonstrated that wide (30 nm) and oxidized and amidated CNTs allow the highest levels of Cas9 conjugation. Cell viability experiments also revealed that incubation of GL261 cells with f-CNTs does not result in cell toxicity. This study established an effective protocol for CRISPR editing in GL261 glioma cells and determined the type of CNT functionalization that allows efficient Cas9 conjugation. For future work, more repeats should be done in order to obtain conclusive results and the cutting and/or knockout efficiency should be assessed with CRISPR-CNTs compared to CRISPR-LipofectAmine.