izpis_h1_title_alt

Razvoj umetnih transkripcijskih dejavnikov na osnovi proteinov Cas9 za regulacijo regulatornih T-celic.
Praznik, Arne (Author), Horvat, Simon (Mentor) More about this mentor... This link opens in a new window

.pdfPDF - Presentation file, Download (4,61 MB)

Abstract
Avtoimunske bolezni, med katere spada tudi diabetes tipa 1, se pogosto razvijejo kot posledica upada mehanizmov imunske tolerance. Pomemben del slednje predstavljajo podskupina T limfocitov, imenovanih regulatorne T celice (Treg), ki s svojo imunosupresivno funkcijo preprečujejo avtoimunsko delovanje lastnih celic in katerih umanjkanje ali nedelovanje je lahko pogosto pomemben faktor v razvoju avtoimunskih bolezni. Za njihov razvoj iz timocitov je ključno stabilno izražanje transkripcijskega faktorja FOXP3, ki deluje kot glavni regulator diferenciacije Treg. Zadostna aktivacija izražanja FOXP3 v drugih T-limfocitih ali celo sorodstveno bolj oddaljenih celicah bi lahko bil zadosten signal za diferenciacijo v Treg in pridobitev imunosupresivnega fenotipa. V magistrskem delu smo poskušali ustvariti in preizkusiti transkripcijske dejavnike, vezane na Cas9 proteine, za specifično aktivacijo izražanja FOXP3. Za usmerjanje Cas9 proteinov na tarčno mesto smo ustvarili različne vodilne RNA (sgRNA) molekule, ki so prepoznale specifična mesta na ključnih regulatornih mestih gena FOXP3. S poskusi in vivo smo dosegli visoko povišanje ob uporabi trodelnega aktivatorja VPR, vezanega na Cas9 protein z izničeno nukleazno aktivnostjo (dCas9:VPR). Najvišjo aktivacijo izražanja FOXP3 smo dobili ob ciljanju promotorske regije gena FOXP3 ter regulatorne regije, katero smo poimenovali Cage1. Po optimizaciji aktivacije izražanja smo prav tako preverili spremembo izražanja ključnih tarčnih genov proteina FOXP3 v celicah, ki imajo FOXP3 utišan. Uvodni poskusi aktivacije izražanja FOXP3 v celični liniji HEK293T so pokazali določeno mero ujemanja izražanja tarčnih genov s Treg celicami. Z nalogo smo pokazali moč metode za tarčno aktivacijo izražanja ter potrdili pomembnost regije Cage1 za tarčno aktivacijo. Metoda bi lahko bila močno orodje pri poskusih diferenciacije celic v Treg, kar bi lahko bilo zlasti pomembno pri terapijah za razne avtoimunske bolezni.

Language:Slovenian
Keywords:CRISPR, dCas9, diabetes tipa 1, FOXP3, regulatorne celice T, plazmidi, HEK293T celice, transfekcija, aktivacija izražanja, regulacija genov, transkripcijski faktorji
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[A. Praznik]
UDC:577.27:606.61:602.6(043.2)
COBISS.SI-ID:8723833 Link is opened in a new window
Views:929
Downloads:593
Metadata:XML RDF-CHPDL DC-XML DC-RDF
 
Average score:(0 votes)
Your score:Voting is allowed only to logged in users.
:
Share:AddThis
AddThis uses cookies that require your consent. Edit consent...

Secondary language

Language:English
Title:Development of Cas9 protein based transcription regulators for the regulation of regulatory T-cells.
Abstract:
Autoimmune diseases, such as type 1 diabetes, often arrise as a result of a defect in one or mechanisms of immune tolerance. An important part of immune tolerance are a subpopulation of T cells, called regulatory T cells (Treg), which prevent the autoimmune activity of aggresive immune cells, and whose depletion or inactivity can lead to severe autoimmune reactions. Stable expression of the master regulator transcription factor FOXP3 is key for their development from thymocytes. Stable induction of FOXP3 expression could potentially drive the differentiation of other T cell population, as well as other cells, towards a Treg lineage and an acquisition of an immosupresive phenotype. In my masters thesis, we aimed at developing and testing transcription activators, bound to modified Cas9 proteins, for the specific induction of FOXP3 expression. We have designed novel short guide RNA (sgRNA), that direct the Cas9 to specific sites on key regulatory regions of the FOXP3 gene. We have achieved high in vivo expression induction of FOXP3 with the use of a tripartite transcription activator VPR, bound to a nuclease-null mutant of Cas9 protein (dCas9:VPR). The highest induction of expression was obtained when we used dCas9:VPR in combination with sgRNA which target the core promotor of FOXP3 and a regulatory region, which we termed Cage1. After the success in achieving transcription activation, we wanted to check the expression profile of key FOXP3 target genes in cells, which normally do not express FOXP3. Initial experiments in HEK293T cell line showed a certain degree of correlation of gene expression to that of Treg cells, after the activation of FOXP3 expression. With our work we have presented a powerfull tool for targeted gene expression, as well as the imporatance of the Cage1 region for the activation of FOXP3 expression. Our tool could be used in further experiments which aim at the generation of Treg cells, which could potentially be used to treat different autoimmune diseases.

Keywords:CRISPR, dCas9, type 1 diabetes, FOXP3, regulatory T cells, plasmids, HEK293T cells, transcription activation, gene regulation, transcription factors

Similar documents

Similar works from RUL:
Similar works from other Slovenian collections:

Comments

Leave comment

You have to log in to leave a comment.

Comments (0)
0 - 0 / 0
 
There are no comments!

Back