In this master's thesis, we attempted to develop and test transcription factors associated with Cas9 proteins for the specific activation of FOXP3 expression, which is crucial for the differentiation of T (Treg) regulatory cells - the task was included in the activities of the 1st set of proposed trials in the project role.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.
F.35 Other
COBISS.SI-ID: 8723833In this bachelor thesis we designed and analysed regulators of the following target genes: LEF1, GATA1, GATA3 and IRF4. Products of these genes, combined with protein Foxp3, are key to maintaining a stable Treg cell phenotype. Modifying expression of these genes can be achieved with the use of the CRISPR/dCas9 system, where regulatory regions of chosen genes act as target sequences for binding of sgRNA, which guides deactivated nuclease dCas9 fused with the activation domain VPR (dCas9-VPR) to the chosen region in the genome. Work was done on Jurkat cells, which were transformed with plasmids, containing target sequences for sgRNA and sequences for the fusion protein dCas9-VPR. We used RT-PCR to determine relative change in the expression of target genes on a transcriptional level. Regulatory regions targeting LEF1 and GATA3 caused a significant change in expression of their target genes. We also confirmed a positive effect of the combination of all four regulators on the expression of gene FOXP3.
F.35 Other
COBISS.SI-ID: 1537596611