Induction of KISS1R expression by CRISPR activation in human embryonic stem cells

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http://urn.fi/URN:NBN:fi:hulib-202207043168
Title: Induction of KISS1R expression by CRISPR activation in human embryonic stem cells
Author: Müller, Linda Helena
Other contributor: Helsingin yliopisto, Bio- ja ympäristötieteellinen tiedekunta
University of Helsinki, Faculty of Biological and Environmental Sciences
Helsingfors universitet, Bio- och miljövetenskapliga fakulteten
Publisher: Helsingin yliopisto
Date: 2022
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202207043168
http://hdl.handle.net/10138/345775
Thesis level: master's thesis
Degree program: Genetiikan ja molekulaaristen biotieteiden maisteriohjelma
Master's Programme in Genetics and Molecular Biosciences
Magisterprogrammet i genetik och molekylära biovetenskaper
Specialisation: Genetiikka ja genomiikka
Genetics and genomics
Genetiikka ja genomiikka
Abstract: Puberty initiation is a crucial physiological process in human development. A group of hypothalamic neurons secreting the gonadotropin-releasing hormone (GnRH) and expressing the kisspeptin receptor (KISS1R) plays a key role in launching puberty. Furthermore, cellular KISS1R signaling has been shown to regulate GnRH expression and secretion. Although the in vitro differentiation of human pluripotent stem cells into GnRH-secreting neurons has been successful, it is of high interest to generate KISS1R expressing GnRH neurons. By utilizing the CRISPR activation technology, this study aimed to establish a conditional KISS1R-activation cell line using H9 human embryonic stem cells. Through controlling dCas9VP192 abundance using the Tet-On system combined with the dihydrofolate reductase destabilizing domain, the transcriptional activation of KISS1R was temporally regulated by the addition of two antibiotic drugs - doxycycline and trimethoprim. KISS1R expression was primarily assessed by qPCR and verified by immunocytochemistry and the use of a KISS1R-GFP reporter cell line. The main finding of this study is the achievement of a 6217 ± 2286 fold change in KISS1R transcription by introducing two guide RNAs (N = 3). Nevertheless, leaky gene activation was observed without drug treatment (fold change of 63 ± 51). Concludingly, this study successfully led to the generation of a KISS1R-activation cell line. After further characterization and refinement of the activation protocol, the established cell line will enable to investigate whether KISS1R upregulation modulates in vitro GnRH neuron differentiation, electrophysiology, hormone expression, and secretion in the future. Respective outcomes may lead to advances in understanding and treating pubertal disorders.
Subject: Puberty
KISS1R
CRISPR activation
human embryonic stem cells


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