University of Twente Student Theses

Login

Insertion of an inducible construct in the genome of human pluripotent stem cells by CRISPR-Cas9 mediated homology directed repair

Hag, G.A. ten (2021) Insertion of an inducible construct in the genome of human pluripotent stem cells by CRISPR-Cas9 mediated homology directed repair.

[img] PDF
5MB
Abstract:Human pluripotent stem cells (hPSCs) have emerged as a promising cells source in cardiac research. The differentiation of hiPSCs towards cardiomyocytes (CMs) and the combination with CRISPR-Cas9 genome editing technology has provided new opportunities for disease modeling, drug screening and tissue engineering. In this research, it was attempted to insert a construct for doxycycline-inducible expression of channelrhodopsin 2 (ChR2) into the AAVS1 genomic safe harbor (GSH) locus of hPSCs by CRISPR-Cas9 mediated homology directed repair (HDR) to enable optogenetic pacing of hPSC-derived CMs (hPSC-CMs). In order to validate transgene expression in the AAVS1 locus of hPSCs and hPSC-CMs, this study showed successful synthetization and characterization of a doxycycline-inducible construct for the expression of the red florescent protein mRuby2. As a proof of concept, hPSCs were co-transfected with the inducible mRuby2 construct and sgRNA-Cas9 plasmid targeting the AAVS1 GSH locus. PCR screening showed successful insertion of the inducible construct in the AAVS1 locus. Surprisingly, doxycycline treatment did not result in mRuby2 signal, which requires further investigation. This study also successfully showed the exchange of the mRuby2 gene with ChR2 coupled to Enhanced Yellow Fluorescent Protein (EYFP) tag (ChR2-EYFP) by sticky-end PCR cloning. It is recommended to start directly with targeting of the inducible construct for ChR2-EYFP expression in hPSC to evaluate transgene insertion and expression, instead of using the mRuby2 construct. Taken together, this study describes the creation of a doxycycline-inducible construct which contains unique restriction sites facilitating the rapid creation a new hPSC line with any genetic alteration of interest involved in cardiac research.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:42 biology, 50 technical science in general
Programme:Biomedical Engineering MSc (66226)
Link to this item:http://purl.utwente.nl/essays/87833
Export this item as:BibTeX
EndNote
HTML Citation
Reference Manager

 

Repository Staff Only: item control page