Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2024. Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj54dlmk/v2Version created by Dan Dou
Manuscript citation:
Pantazis, C.B., Yang, A., Lara, E., McDonough, J.A., Blauwendraat, C., Peng, L., Oguro, H., Zou, J., Sebesta, D., Pratt, G., et al. (2022). A reference induced pluripotent stem cell line for large-scale collaborative studies. BioRxiv 2021.12.15.472643.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
We adapted a previously-described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.”
Wear proper PPE when transferring cryovials to liquid N2.
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
3d 6h
Culture iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel (Corning) and feed daily with Essential 8 media (ThermoFisher).
Passage iPSCs with warm Accutase into Essential 8 media with 10 micromolar (µM) ROCK inhibitor. Plate 800,000 iPSCs into one Matrigel-coated well of a 6-well plate.
3 - 6 hours after plating, cells should be healthy and attached. Perform transfection using Lipofectamine Stem and a 2:1 ratio of donor plasmid to transposase:
A
B
OptiMEM
200 μL
PB-TO-hNGN2-puro-BFP plasmid
0.75 μg
EF1α-transposase plasmid
0.37 μg
Lipofectamine Stem
4 μL
Check for transfection efficiency (BFP-labeled cells) on the next day using fluorescence microscopy.
Passage iPSCs with Accutase to a 10 cm dish when cells are confluent enough for splitting.
72:00:00 after transfection, select for transfected iPSCs with 0.5 Mass Percent puromycin.
3d
Confirm purity of surviving transfected cells with fluorescence microscopy. When population is pure, withdraw puromycin.
Cryopreserve selected iPSCs with
A
B
Essential 8 media
70%
Knockout serum replacement
20%
DMSO
10%
ROCK inhibitor (Supplement)
10 µM
Proceed to culture and induction to neuronal fate using doxycycline (see “Protocol: iNeuron differentiation from human iPSCs”).