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The role of celiac desease associated genes in T cell activation

Dekker, B (2015) The role of celiac desease associated genes in T cell activation. Master's Thesis / Essay, Biology.

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Many mutations and genetic loci that have been identified in genome wide association studies (GWAS) are shared between different autoimmune diseases, and many potentially causal genes seem to have a role in immune response, i.e. T-cell differentiation and signaling. Examples of these genes that have a putative function in immune response are UBE2L3, UBASH3A and long non-coding RNA (lncRNA) AC104820.2. In thesis we will look in further detail where these three genes may play a role in T cell activation and more importantly where they may play a role in the pathogenesis of celiac disease. In order that Crispr/Cas9 was used to generate knock out cell lines in Jurkat and Hek293T cells. Jurkat cells are notorious difficult to transduce and therefore transduction optimization was needed. Resulting in transduction efficiencies increased up to ~60%. Furthermore, GFP was cloned into the pLentiCrisprV2 vector to enable positive selection after the transduction, instead of previously used negative selection. Both the enhanced transduction efficiency and GFP expression increase the chance to obtain cell lines with knock outs. However, for Jurkat cell there is yet no clone found although this seems a matter of time looked at the Crispr/Cas9 efficiency. For Hek293T various UBE2L3 knock down cell lines were generated and functionally tested. Stimulation upon TNF-α showed almost no different expression as the wild type. Only the most severe knock down clones had increased IκBα and Il-8 expression. This was unexpected but yet very interesting.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Biology
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 08:08
Last Modified: 15 Feb 2018 08:08

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