Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain

Current Protocols in Stem Cell Biology
Aslam Abbasi Akhtar, Joshua J Breunig

Abstract

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.

References

Apr 22, 2003·Nature Neuroscience·Alan CarletonPierre-Marie Lledo
Nov 18, 2008·Developmental Biology·Mitsuru Morimoto, Raphael Kopan
Apr 21, 2011·Nature Reviews. Neuroscience·Jessica L AblesPasko Rakic
Apr 24, 2012·Journal of Neuroscience Methods·Fuyi Chen, Joseph LoTurco
Sep 21, 2013·Stem Cell Reports·Simon M G BraunSebastian Jessberger
Feb 11, 2014·Neuron·Karine LoulierJean Livet
May 20, 2014·Journal of Visualized Experiments : JoVE·Rachelle LevyJoshua J Breunig
Jul 3, 2015·Frontiers in Cellular Neuroscience·Aslam Abbasi Akhtar, Joshua J Breunig
Jul 7, 2015·Cell Reports·Joshua J BreunigMoise Danielpour
Apr 14, 2016·Molecular Therapy. Methods & Clinical Development·Abdelwahed ChtartoLiliane Tenenbaum

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