Masters Thesis

Disruption of Human Transmembrane (tmem)-176a and -176b Expression Using Rna Interference and Crispr/cas9 Technology

Transmembrane (TMEM)-176A and -176B proteins have been reported to play a role in human immune cell function. Similarity in structure, high homology, and a close genetic relationship enable the two proteins to physically come together to form heteromeric and homomeric multimers. A recent study in rodent immune cells discovered the ability of Tmem176A and -176B to come together to form functional ion channels near the trans-Golgi Network (TGN). Here, they may influence intracellular signaling events that could ultimately be regulating immune functions. Abnormal accumulations of such multimers have been found to restrain dendritic cell maturation and activation. Therefore, TMEM176A and -176B may serve as potential targets for immune cell regulation. Additionally, increased protein levels of TMEM176A and -176B have been linked to cancer pathology. However, the specific role of these two proteins as they relate to immune tolerance and cancer pathology remains to be elucidated. We hypothesize that TMEM176A and -176B are involved in the evasion of immune surveillance of cancer cells. This study was aimed at creating TMEM176A and -176B knockdown cell lines for use as culture models in future studies. To this end, RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats with Cas9 nuclease (CRISPR/Cas9) techniques were used to identify the most efficient means to knock down the endogenous expression of TMEM176A or -176B. Subsequently, stable knockdown iii cell lines were created. RNAi did not significantly or consistently reduce the expression of TMEM176A or -176B in HEK293T cells. CRISPR/Cas9, however, successfully conferred mutations in the targeted regions of TMEM176A and -176B in three stable HEK293T cell lines using pX459-TMEM176A gRNA1 or pX-TMEM176B gRNA1. However, qualitative analysis revealed only a subsequent reduction of TMEM176A expression in two cell lines generated by pX-TMEM176A gRNA1 transfection. Therefore, pX459-TMEM176A gRNA1 is the best candidate for CRISPR/Cas9-mediated knockdown in HEK293T cells. These culture models, if used in future studies, could shed additional light on the role of the two TMEM176 proteins in evasion of immune surveillance of cancer cells.

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