<b>The Role of Circular RNA in Adaptive Immune Cells</b>

<p dir="ltr">Circular RNAs (circRNAs) are a class of noncoding RNAs (ncRNAs) that can regulate gene expression through different mechanisms, such as inhibiting or “sponging” microRNA (miRNA), thus modulating miRNA targets. CircRNA function has been increasingly recognized in cancer but remains less characterized in adaptive immune cells. This thesis focused on developing approaches to investigate the function of circRNAs in human CD4+ T cells, with an emphasis on Th1 differentiation and IFNγ production.</p><p dir="ltr">To test the functionality of siRNA/shRNA-mediated knockdown of circRNAs in primary human T cells, circSATB1 was knocked down in Treg-differentiated cells. With decreased expression of circSATB1 after knockdown, siRNA or shRNA is a feasible tool for RNA interference. With that confirmation, a pooled short hairpin RNA (shRNA) library was designed to target the back-splicing junction (BSJ) sites of2,000 circRNAs previously identified in CD4+ T cells. Construction of this library required extensive optimization, including amplification of shRNA inserts, linearization of the shRNA vector, and In-Fusion cloning. Several experimental parameters were tested, such as PCR cycling conditions, annealing temperature, and the inclusion of additives like betaine. Despite these optimizations, overall shRNA library coverage remained below the desired threshold of 30X, reflecting the technical challenges of constructing highly complex circRNA-targeting libraries.</p><p dir="ltr">Together, this work provides a method for developing a circRNA-targeting shRNA library construction and highlights important considerations for its optimization. These findings lay the groundwork for future functional screening of circRNAs in adaptive immune cells and for understanding their potential regulatory roles in T cell differentiation and cytokine production</p>