Testiranje sistema CRISPR/Cas9 za razvoj mišjega modela za avtizem

Štravs, Ana (avtor), Dovč, Peter (mentor), Horvat, Simon (komentor)

Motnje avtističnega spektra (MAS) so skupina heterogenih nevroloških motenj, ki se razvijejo v zgodnjem otroštvu in pri katerih ima genetsko ozadje močan vpliv. Zaradi visoke genetske variabilnosti in kombinacij faktorjev, ki prispevajo k nastanku bolezni, je mehanizem le-te težko pojasniti. Napredek v humani genetiki in funkcionalni genomiki je izpostavil veliko potencialnih kandidatnih genov, katerih vlogo je potrebno preučiti. V diplomski nalogi smo raziskali potencialni kandidatni gen za avtizem astrotaktin 2 (ASTN2, angl. astrotactin 2) in na podlagi literature opisali njegovo vlogo pri nevralnih migracijah v malih možganih kot prispevajoč faktor za razvoj MAS. Testirali smo potencial CRISPR/Cas9 sistema za razvoj mišjega modela za MAS, s katerim bi lahko v bodoče izvedli obsežno analizo in tako določili povezave med molekularnimi, fiziološkimi, funkcionalnimi in vedenjskimi faktorji. S preliminarnimi poskusi in vitro in in vivo smo poskušali opredeliti izvedljivost uporabe tega pristopa za razvoj mišjega modela. Za tarčo smo si izbrali ekson 2 mišjega gena astrotaktina 2, Astn2. Primernost izbrane sgRNA smo potrdili z in vitro cepitvijo in testom s sistemom pCAG-EGxxFP pri celični liniji HEK293. Rezultati diplome so pokazali, da je izbrana sgRNA učinkovita in robustna za uporabo v nadaljnjih poskusih in vitro in in vivo.

• Jezik: Slovenski jezik
• Ključne besede: biotehnologija, CRISPR/Cas9, motnje avtističnega spektra, astrotaktin 2, mišji model
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: BF - Biotehniška fakulteta
• Založnik: [A. Štravs]
• Leto izida: 2019
• PID: 20.500.12556/RUL-107883
• UDK: 60:616.896(043.2)
• COBISS.SI-ID: 9233529
• Datum objave v RUL: 02.06.2019

ŠTRAVS, Ana, 2019, Testiranje sistema CRISPR/Cas9 za razvoj mišjega modela za avtizem [na spletu]. Diplomsko delo. A. Štravs. [Dostopano 3 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=107883

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Testing of CRISPR/CaS9 system for development of mouse model for autism spectrum disorders

Izvleček

Autism spectrum disorders (ASD) is a group of early onset heterogenous neurological disorders with strong genetic predisposition. Due to very high genetic and phenotypic variability and combinations of factors contributing to disease onset, elucidating the mechanism of the disorder is in most cases difficult. Advances in human genetics and functional genomics have brought to light many potential candidate genes, whose roles are yet to be determined. We investigated an ASD candidate gene astrotactin 2 (ASTN2) as a potential risk gene and based on the literature review described its role in cerebellar neural migration as a possible contributing factor for ASD. We tested the potential of CRISPR/Cas9 technology for development of a mouse model of ASD, with which we could in future conduct functional in vivo analyses to determine the associations between molecular, physiological, functional and behavioural factors. This study consisted of preliminary molecular experiments in vitro and in vivo, to determine the feasibility of using this approach to generate a murine Astn2 gene knockout model. We chose the mouse Astn2 exon 2 as our target site for sgRNA and Cas9 cleavage. We confirmed the efficiency of this sgRNA for gene editing using in vitro DNA cleavage assay and GFP-based detection system in the HEK293 cell line by employing the pCAG-EGxxFP plasmid. We concluded that the selected sgRNA is robust and effective for use in further functional experiments in vitro and in vivo.

• Ključne besede: biotechnology, CRISPR/Cas9, autism spectrum disorders, astrotactin 2, murine model