Browsing by Subject "IER3IP1"

IER3IP1 is a protein located in the endoplasmic reticulum (ER) transmembrane, and it is highly expressed in pancreatic beta cells and developing brain cortex. The loss-of-function mutations in IER3IP1 cause monogenic neonatal diabetes together with brain linked diseases such as epilepsy and microcephaly. The aim of this thesis is to study the role of IER3IP1 in the development and function of human beta cells using hESC-derived pancreatic islets. Using CRISPR/Cas9, IER3IP1 knockout (KO) and IER3IP1 loss-of-function mutation knock-in (KI) hESC clones were generated. For KO, the first exon of IER3IP1 was deleted whereas for KI, the 21. valine of IER3IP1 was changed to glycine. The clones together with their unedited controls (H1), were differentiated into pancreatic stem cell (SC)-islets following the optimized 7-stage differentiation protocol. The differentiation was followed during the protocol and the SC-islets were tested at the end of the protocol. In vitro, IER3IP1 KO-islets contained less beta cells and more alpha cells when compared to the H1-islets, as shown by immunostainings for insulin and glucagon. The beta cells of KO-islet accumulated more proinsulin compared to H1-islets and had significantly higher level of ER-stress shown by elevated ER-stress marker BiP. Moreover, the KO-islets showed drastically lower amount of insulin secretion and diminished insulin content. The IER3IP1 KI-islets did not significantly differ from H1-islets. Thus, this master’s thesis shows that IER3IP1 is essential for maintaining normal ER homeostasis and beta cell function in vitro. In future, these results should be confirmed using in vivo model.