Aktivacija izražanja gena Tst z metodo CRISPR/dCas9-VPR v mišji celični liniji

Lenardič, Ajda (author), Horvat, Simon (mentor)

Debelost in z njo povezana sladkorna bolezen tipa 2 (SBT2)sta med najbolj razširjenimi boleznimi sodobnega časa. Na svetu je 13% odraslih debelih in 39 % prekomerno težkih, za SBT2pa trpi okrog 8 % svetovne populacije. Zaenkrat še ne poznamo učinkovite terapije, ki bi uspešno zdravila ali celo preprečevala obe bolezni, hkrati pa ne bi imela sistemskih oziroma centralnih učinkov.Tst kot eden redkihgenovz dokazano vlogo pri ohranjanju metabolično zdravega fenotipa predstavlja potencialno zelo zanimivo terapevtsko tarčo. Na podlagi predhodno dokazane korelacije med visokim izražanjem Tst in nizkimdeležemmaščevjaternizkim deležem plazemske glukozepri miših in ljudeh smo v diplomskinalogi s sistemom CRISPR/dCas9-VPR poskušali povečati izražanje Tstv trajni celični linijimišjega izvora,NIH3T3.Ta celični model bilahkoslužil kot osnova za nadaljnje raziskovanje in razvoj aplikacij, na primer terapij za zdravljenje SBT2 in debelosti. Potencialno tarčno regijo za vezavo sgRNA na lokusuTst smo izbrali na osnovi bioinformacijskeanalize regulatornih elementov tarčnega gena. Glede na izbrano tarčnoregijosmo zasnovali sgRNA.S cepitvijo DNA in vitro in s testomz vektorjem pCAG-EGxxFP v celicah HEK293 smo dokazali, da se izbrana sgRNA ustrezno veže na tarčno DNA tako in vitro kot v celičnem okolju. Z uspešnim povečanjem izražanja gena Oct4 smo preverili tudi, da proteinska fuzija dCas9-VPR v celicah NIH3T3 deluje ustrezno.Skombinacijo zasnovane sgRNA in proteinske fuzije dCas9-VPR iz vektorja pCMV-dCas9:NLS:VPRsmo natoposkušali povečati izražanje Tst v celicah NIH3T3, vendar statistično značilnega povečanjatranskripcije nismo opazili.Najverjetnejšivzroki za to so prenizka učinkovitost transfekcije, neustrezno izbrana tarčna regija za vezavo sgRNA, odsotnost konstitutivnega izražanja sgRNA zaradi vnosa slednje v sintetični obliki ali pa odsotnostdoločenega transkripcijskega dejavnika, ki je potreben za izražanje gena Tst,vcelicah NIH3T3.Kljub temu, da izražanja Tst nismo uspeli statistično značilnopovečati, smo v diplomskem delu prišli do nekaterih ugotovitev, ki bi lahko služile kot osnova za nadaljnje poskuseregulacije izražanja genov s sistemom CRISPR/dCas9 in s tem pri razvoju terapij za debelost, SBT2 indruge bolezni, ki bi jih bilomogoče zdraviti z regulacijo izražanja endogenih genov.

• Language: Slovenian
• Keywords: CRISPR/dCas9-VPR, Tst, NIH3T3, debelost, sladkorna bolezen tipa 2
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-113450
• COBISS.SI-ID: 1538397123
• Publication date in RUL: 08.01.2020

Secondary language

• Language: English
• Title: Tst gene expression activation by using CRISPR/dCas9-VPR system in mouse cell line

Abstract

Nowadays, obesity and type 2 diabetes, often associated with it,are amongst the most widespread health disorders. According to the World Health Organisation, 13 % of the world adult population is obese and 39 % is overweight;8 %suffer from type 2 diabetes. Currently, treatment that would efficiently cureor even prevent both diseases without having systemicand/orcentral effects simultaneouslydoes not exist. Being one of a fewgeneswith the proven role in maintaining metabolic health, Tst manifestsa very interestingpotential therapeutic target. High Tst expressionhas been proven tocorrelate withlow fat massand with low blood glucose levels in mice as well as in humans. Based on thesecorrelations, we triedto activate Tst expression in mouse cell line NIH3T3by using CRISPR/dCas9-VPR system. Firstly,target DNA sequence for sgRNA binding was chosenbased on the regulatory regions in Tst locus. According tothe chosen target sequence, sgRNA was designed. By successful cleavage of the target DNA in vitro and in the cellular environment, we showedthat designed sgRNA is able to bind target DNA sequence. By using activationcomplex dCas9-VPR, expressed from vector pCMV-dCas9:NLS:VPR, wesuccessfully activated Oct4 transcription and therefore provedthat this activationcomplex works well in NIH3T3 cell line.Having both components tested, we tried to activate transcription of Tst by transfecting NIH3T3 cells with thedesigned sgRNA and vectorpCMV-dCas9:NLS:VPR. Unfortunately, nosignificant change in gene expressionwas observed. Lack of activation mighthavebeenthe consequence ofvery poor transfection efficiency and/or wrong choice of the target DNA region and/orlack of constitutive expression of sgRNA, since the cellswere transfected with sgRNA in synthetic formand/or lack of transcription factor(s) required for Tst expression in NIH3T3cell line.DespiteunsuccessfulTstactivation,we believe that our work providesgood basis for additional attempts of Tst–andpotentially some other gene –expression activation using CRISPR/dCas9 systemand therefore for the developmentof treatments for obesity,type 2diabetes and some other diseases that couldbe treated by genetic manipulations.

• Keywords: CRISPR/dCas9-VPR, Tst, NIH3T3, obesity, type 2 diabetes