The role of CD3 delta and Rag1Ap1 proteins in T cell receptor expression
Keskin, Nazlı (2010) The role of CD3 delta and Rag1Ap1 proteins in T cell receptor expression. [Thesis]
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CD3 subunits of the of T cell receptor (TCR) are essential for the assembly and signal transduction initiated from this receptor complex in T lymphocytes. The first part of this study aims to generate a CD3 delta conditional knockout construct by recombineering bacterial artificial chromosomes. We generated multiple plasmids that were designed to target the CD3 delta gene locus in mouse embryonic stem cells. In the second part of the study, we focused on a CD3 delta interacting protein called Rag1Ap1, which was identified in a yeast two hybrid screen. We confirmed the interaction of CD3 delta and Rag1Ap1 by co-immunoprecipitation in HEK 293T cells. To study the subcellular localization of the CD3 – Rag1Ap1 interaction, we generated a plasmid expressing a Rag1Ap1 – Venus (YFP) fusion protein. We analyzed the subcellular localization of the Rag1Ap1-Venus protein by fluorescent and confocal microscopy in transfected HeLa cell lines. Furthermore, we tested the effect of the overexpression and shRNA mediated knockdown of Rag1Ap1 on TCR expression levels in T lymphocyte cell lines, by flow cytometry. These experiments indicate that Rag1Ap1 interacts with CD3 delta in the endoplasmic reticulum and may play a role in TCR assembly and surface expression.
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