Open AccessTetracycline‐Inducible and Reversible Stable Gene Expression in Human iPSC‐Derived Neural Progenitors and in the Postnatal Mouse Brain
Author(s) -
Akhtar Aslam Abbasi,
Breunig Joshua J.
Publication year - 2017
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/cpsc.28
Subject(s) - nucleofection , biology , reporter gene , transgene , microbiology and biotechnology , neural stem cell , progenitor cell , induced pluripotent stem cell , luciferase , bioluminescence imaging , transfection , cell culture , gene , gene expression , stem cell , embryonic stem cell , genetics
The pB‐tet‐GOI plasmid system allows for stable piggyBac transposition‐mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non‐invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. Specifically, a detailed protocol is provided to illustrate: (1) cloning of the respective GOI (genetic element(s) of interest); (2) nucleofection of the plasmid system into human induced pluripotent stem cell (iPSC)‐derived neural progenitors; (3) dox‐induced activation in vitro or in vivo; and (4) non‐invasive assessment of gene activity in vivo by bioluminescence imaging. © 2017 by John Wiley & Sons, Inc.