Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging

C H Arendzen; U Chaudhari; S J Cramer; C M A H Freund; C L Mummery; A Ranga; O Pourquie; H M M Mikkers

doi:10.1016/j.scr.2021.102592

Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging

Stem Cell Res. 2021 Dec:57:102592. doi: 10.1016/j.scr.2021.102592. Epub 2021 Nov 3.

Authors

C H Arendzen¹, U Chaudhari², S J Cramer³, C M A H Freund², C L Mummery², A Ranga⁴, O Pourquie⁵, H M M Mikkers⁶

Affiliations

¹ LUMC hiPSC Core Facility, Leiden University Medical Center, Netherlands; Dept of Anatomy and Embryology, Leiden University Medical Center, Netherlands. Electronic address: [email protected].
² LUMC hiPSC Core Facility, Leiden University Medical Center, Netherlands; Dept of Anatomy and Embryology, Leiden University Medical Center, Netherlands.
³ Dept of Cell and Chemical Biology, Leiden University Medical Center, Netherlands.
⁴ Dept of Biomechanics, KU Leuven, Belgium.
⁵ Harvard Stem Cell Institute, Harvard Medical School, USA.
⁶ LUMC hiPSC Core Facility, Leiden University Medical Center, Netherlands; Dept of Cell and Chemical Biology, Leiden University Medical Center, Netherlands. Electronic address: [email protected].

PMID: 34775202
DOI: 10.1016/j.scr.2021.102592

Abstract

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.

Keywords: Cell-Cylce Reporter; Histon2B; hiPS cell line.