Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.
Publication Year:
2023
PubMed ID:
37283517
Funding Grants:
Public Summary:
Our team created methods to safely and stably add special genes into mammalian cells without using viruses. Using a system called piggyBac, we can insert genes that can be turned on or off by adding a drug called doxycycline. We track which cells have these genes by making their nuclei glow, and we measure gene activity non-invasively with a light-producing marker called luciferase. We also developed a newer system called MADR for similar gene control. These methods work both in lab-grown cells and in young mouse brains, and we provide step-by-step instructions for cloning, inserting, activating, and measuring these genes.
Scientific Abstract:
Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the 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. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. (c) 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.