Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4

master's thesis

• Title (english): Epigenetic manipulation of the MGAT4A and MGAT5 genes in pancreatic model cell line 1.1B4 using VPR-dCas9 molecular tool
• Author: Dora Lalić
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-10-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Transporteri GLUT (engl. Glucose Transporter) nalaze se na membranama beta-stanica gušterače
te su odgovorni za unos glukoze iz krvotoka, što u konačnici dovodi do ispuštanja inzulina koji
regulira razinu glukoze u krvi. U miševa je dokazano da nedostatak glikoziltransferaza GnT-4a i
GnT-V, kodiranih genima MGAT4A i MGAT5, uzrokuje nepravilnu glikozilaciju transportera
GLUT i smanjenje njihove količine u membranama, što posljedično dovodi do razvitka dijabetesa
tipa 2. Čini se da isti mehanizam postoji i u ljudima, ali mreža molekularnih događaja uključenih
u nepravilnu regulaciju glikoproteina GLUT nije još razjašnjena.
U ovom je istraživanju ciljano povećana ekspresija gena MGAT4A i MGAT5 pomoću
molekularnog sustava CRISPR/dCas9 koji na sebe ima fuzioniranu aktivacijsku domenu VPR
kako bi se ispitao učinak na količinu i lokalizaciju transportera GLUT1 u stanicama 1.1B4, koje
su model za ljudske beta-stanice gušterače. Ekspresija gena uspješno je povećana na razini
transkripcije te je potvrđena reverznom transkripcijom i lančanom reakcijom polimeraze u
stvarnom vremenu (qPCR). Metodom Western blot analizirana je količina proteina GnT-4a i GnTV te je uočen porast količine koji nije statistički značajan. Imunofluorescencijskim bojanjem
analizirana je količina i lokalizacija transportera GLUT1 te je uočen gotovo dupli porast u količini
transportera između tretiranih i kontrolnih stanica. Kako bi se istražilo na koji je način povećana
količina enzima utjecala na povećanje količine transportera GLUT1, trebalo bi analizirati glikanski
profil transportera.

Abstract (english)

GLUT transporters (Glucose Transporters), localized in membranes of pancreatic beta cells, are
responsible for uptake of glucose from the bloodstream. That leads to release of insulin which
regulates blood sugar level. It has been proven in mice that the absence of glycosytransferases
GnT-4a and GnT-V, encoded by genes MGAT4A and MGAT5, leads to improper glycosylation of
GLUT transporters and a decrease in their abundance, which leads to development of type 2
diabetes. It appears the same mechanism exists in people, however the network of exact molecular
events that lead to impaired glycosylation of GLUT transporters has not yet been resolved.
In this study, expression of genes MGAT4A and MGAT5 was upregulated using a CRISPR/dCas9
system to which an activating VPR domain was fused. Cell line 1.1B4 was used, which is a model
cell line for human pancreatic beta cells, so the effect on GLUT transporters could be investigated.
Gene upregulation was confirmed on a transcription level by reverse transcription and quantitative
polymerase chain reaction (qPCR). Protein analysis was done by Western blot and an increase in
protein quantity was observed, but it was statistically unsignificant. Imunofluorescent staining was
used to analyse the quantity and localization of GLUTs. Treated cells had almost double the
quantity of GLUTs compared to the control group. To investigate the mechanism by which
elevated levels of GnT-4a and GnT-V affected on GLUT quantity, glycan profile of transporters
would have to be analysed.

• Keywords: CRISPR/dCas9; MGAT4A; MGAT5; dijabetes; GLUT1; stanična linija 1.1B4
• Keywords (english): CRISPR/dCas9; MGAT4A; MGAT5; diabetes; GLUT1; 1.1B4 cell line
• Language: croatian
• URN:NBN: urn:nbn:hr:217:058743
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-11-17 11:31:11