Optimizacija elektroporacije za aktivacijo genov vključenih v diferenciacijo regulatornih celic T

Martinšek, Klemen (author), Oven, Irena (mentor)

Regulatorne celice T so specializirana podpopulacija limfocitov T, ki s svojim delovanjem vzdržujejo imunsko toleranco. Odsotnost regulatornih celic T vodi v razvoj avtoimunskih obolenj in v nenadzorovano proliferacijo limfocitov T in B, kar vodi v nastanek kroničnih vnetij. Dokazano je, da z vnosom regulatornih celic T stanje bolezni lahko izboljšamo, zato so v razvoju metode za diferenciacijo konvencionalnih limfocitov T v regulatorne limfocite T. V diplomskem delu smo v Jurkat celice z elektroporacijo vnesli dCas9-VPR in različne kombinacij plazmidov za sgRNA ter s tem poskusili aktivirati za diferenciacijo pomembne gene. Merili smo spremembo ekspresije genov FOXP3, CTLA4, EOS, CD25 in CD4. Najvišje povišanje izražanja FOXP3 smo dosegli s kombinacijo koaktivacije genov FOXP3 in CTLA4. Slednja kombinacija je bila najuspešnejša tudi v primeru zvišanja ekspresije CD25. Ekspresija CD4 je ostala v večini primerov nespremenjena. Kostimulacija genov FOXP3, CTLA4 in EOS je v nasprotju s pričakovanji pokazala na negativen učinek na ekspresijo opazovanih genov. V diplomskem delu smo dokazali, da kombinacija vnesenih plazmidov vpliva na različno ekspresijo genov pomembnih za diferenciacijo regulatornih celic T.

• Language: Slovenian
• Keywords: biotehnologija, eletroporacija, regulatorne celice T, avtoimune bolezni, CRISPR-Cas, FOXP3, CTLA4, EOS, CD4, CD25
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [K. Martinšek]
• Year: 2018
• PID: 20.500.12556/RUL-102608
• UDC: 602.621:602.68:606:61(043.2)
• COBISS.SI-ID: 9058425
• Publication date in RUL: 05.09.2018

Secondary language

• Language: English
• Title: Optimization of electroporation of genes involved in activation of differentiation of regulatory T cells

Abstract

Regulatory T cells are specialized subpopulation of lymphocytes T, which areresponsible for maintaining the immune tolerance. The absence of TREG can lead to development of autoimmune diseases and to uncontrolled proliferation of lymphocytes T and B, which in result can lead to chronic inflammation. It has been proven that the condition of the disease can be improved with the introduction of new TREG. Scientists are trying to develop a method for differentiation of conventional T cells into TREG. In the diploma, we tried to activate the genes involved in differentiation into TREG, by electroporating dCas9-VPR and various combinations of plasmids for sgRNAs into Jurkat cells. We measured the change in the level of expression of FOXP3, CTLA4, EOS, CD25, and CD4 genes. The highest increase in expression of FOXP3 was achieved by co-activation of FOXP3 and CTLA4 genes. The latter was also the most successful in increasing CD25 expression. The CD4 expression remained unchanged. Contrary to expectations, co-activation of FOXP3, CTLA4 and EOS genes showed a negative effect on expression of observed genes. In the diploma, we have shown that the combination of electroporated plasmids affects the differential expression of genes important for the differentiation of TREG.

• Keywords: biotechnology, electroporation, regulatory T cells, autoimmune diseases, CRISPR-Cas, FOXP3, CTLA4, EOS, CD4, CD25