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CRISPR/Cas9-Mediated Activation of FOXL2 in Human Pluripotent Stem Cells

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Title: CRISPR/Cas9-Mediated Activation of FOXL2 in Human Pluripotent Stem Cells
Author(s): Nedeczey-Ruzsák, Petra Dalma
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences
Degree program: Master 's Programme in Neuroscience
Specialisation: Neuroscience
Language: English
Acceptance year: 2023
Normal sex differentiation depends largely on the healthy development of the bipotential gonad, which is identical in both sexes during early stages of embryonic development. Sex differentiation towards the female phenotype is initiated by the expression of pro-ovarian genes, among which Forkhead Box L2 (FOXL2) is an important regulator. Moreover, FOXL2 was found to be one of the genes most widely implicated in female disorders of sex development (DSD). However, there is a lack of understanding regarding its precise role during ovarian differentiation and development. In order to study the gene during early gonadal development, human embryonic stem cells (hESCs) were used as a model. An inducible FOXL2 activation line was generated in vitro, by applying the CRISPR/Cas9 technique in combination with the tetON and destabilized DHFR systems. The cells were also subjected to gonadal differentiation, based on a previously established protocol. The results showed that the establishment of the activation line was successful, and expression of FOXL2 could only be observed in cells that were treated with trimethoprim and doxycycline. Similar findings were observed in the differentiated activator cells, as again only the induced cells expressed FOXL2. On the other hand, both induced and non-induced differentiated cells showed expression of bipotential gonadal marker genes LHX9, EMX2, GATA4 and WT1. However, in the induced cells a lower relative expression of these markers could be observed. Therefore it seems that relative expression of bipotential gonadal markers was affected by FOXL2 activation. The expression of female gonadal marker genes RSPO1, FSHR, WNT4, AMH and FST was not influenced by FOXL2 activation during gonadal differentiation, as most of the markers showed similar levels of expression in both induced and non-induced cells. Therefore further research needs to be conducted to determine optimal time point of FOXL2 activation during differentiation. Nevertheless, an in vitro model could be generated, which could help in the future to further study the role of FOXL2 in gonadal differentiation, and to better understand pathological mechanisms underlying female DSDs.
Keyword(s): Gonadal Development FOXL2 Disorders of Sex Development hESCs CRISPR/dCas9 tetON

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