Browsing by Subject "gonadal development"

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is a master regulator of both steroidogenesis and gonadal development. Disruptions of NR5A1 can result in differences in sexual development (DSD). With proven interspecies differences in NR5A1 functioning and human material not being available, human stem cells are one of the most achievable, ethical, and accurate models to study the earliest developmental stages of foetal life. However, in currently existing human stem cell-derived gonadal models the expression of NR5A1 has been insufficient without artificial induction due to the lack of knowledge of its distinct biological mechanisms, endogenous ligands, and co-factors. A functional reporter cell line would enable high throughput microscope screening of differentiation protocols with expressed NR5A1. The aim of this thesis was to generate a functional monoclonal human embryonic stem cell (hESC) reporter line for the gene NR5A1 with Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) complex. Firstly, an efficient guide RNA was determined for NR5A1 by T7 assay, and a homology-directed repair (HDR) donor plasmid was designed based on it. Secondly, monoclonal hESC lines were generated with the Alt-R CRISPR-Cas9 RNP complex knock-in method and HDR donor plasmid via electroporation and single-cell sorting. Finally, monoclonal hESC reporter lines were screened with Touchdown PCR and a functionality analysis based on fluorescence and mRNA expression was performed. Two monoclonal hESC reporter lines H9-NR5A1-eGFP cl. 1 and dual-inducible H9-NR5A1-DDdCas9VP192-eGFP cl. 28 were established by using Alt-R CRISPR-Cas9 RNP complex. However, a functional validation performed on H9-NR5A1-DDdCas9VP192-eGFP cl. 28 cells showed the cell line to be non-functional upon NR5A1 upregulation regardless of the expressed eGFP mRNA detected with RT-qPCR.