PiggyBac Transposon-Mediated Gene Editing in Human iPSCs: A Procedure to Integrate Gene of Interest in Human iPSCs Using PiggyBac Transposon System

Published: April 30, 2023

Abstract

Source: Garone, M. G. et al. Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors. J. Vis. Exp. (2019)

This video demonstrates the procedure to generate motor neurons by transfection of human iPSCs with PiggyBac transposon system by the ectopic expression of lineage-specific transcription factors.

Protocol

1. Generation of NIL and NIP Inducible iPSC Lines Cell transfection Rinse the cells with PBS (Ca2+/Mg2+ free). Add cell dissociation reagent (see Table of Materials) (0.35 mL for a 35 mm dish) and incubate at 37 °C until single cells are separated (5-10 min). Gently complete cell separation by pipetting up and down with a P1000 pipettor 3-4 times. Collect in a 15 mL tube and ad…

開示

The authors have nothing to disclose.

Materials

B27 Miltenyi Biotec  130-093-566 Serum free supplement for neuronal cell maintenance
Bambanker Nippon Genetics NGE-BB02 Cell freezing medium for motor neuron progenitors
CaCl2 Sigma-Aldrich C3881 Chemicals for electrophysiological solutions
Corning Matrigel hESC-qualified Matrix Corning 354277 Reconstituted basement membrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Used for adhesion of iPSC to plastic and glass supports
CRYOSTEM ACF FREEZING MEDIA Biological Industries 05-710-1E Freezing medium for human iPSCs
Dispase Gibco 17105-041 Reagent for gentle dissociation of human iPSCs
DMEM/F12 Sigma-Aldrich D6421-500ML Basal medium for cell culture
Doxycycline Sigma-Aldrich D9891-1G Used to induce expression of transgenes from epB-Bsd-TT-NIL and epB-Bsd-TT-NIP vectors
DS2U WiCell UWWC1-DS2U Commercial human iPSC line
GDNF PreproTech 450-10 Glial-Derived Neurotrophic Factor
Gibco Episomal hiPSC Line Thermo Fisher Scientific A18945 Commercial human iPSC line
Hepes Sigma-Aldrich H4034 Chemicals for electrophysiological solutions
Laminin Sigma-Aldrich 11243217001 Promotes attachment and growth of neural cells in vitro
NEAA Thermo Fisher Scientific 11140035 Non-Essential Amino Acids. Used as a supplement for cell culture medium, to increase cell growth and viability.
Neon 100 μL Kit Thermo Fisher Scientific MPK10096 Cell electroporation kit
Neon Transfection System Thermo Fisher Scientific MPK5000 Cell electroporation system
Neurobasal Medium Thermo Fisher Scientific 21103049 Basal medium designed for long-term maintenance and maturation of neuronal cell populations without the  need for an astrocyte feeder layer
NutriStem-XF/FF Biological Industries 05-100-1A Human iPSC culture medium
PBS Ca2+/Mg2+ free Sigma-Aldrich D8537-500ML Dulbecco's Phosphate Buffer Saline, w/o Calcium, w/o Magnesium
Penicillin/Streptomycin Sigma-Aldrich P4333-100ML Penicillin/Streptomicin solution used to prevent cell culture contamination from bacteria.
Y-27632 (ROCK inhibitor) Enzo Life Sciences ALX-270-333-M005 Cell-permeable selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Increases iPSC survival

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記事を引用
PiggyBac Transposon-Mediated Gene Editing in Human iPSCs: A Procedure to Integrate Gene of Interest in Human iPSCs Using PiggyBac Transposon System. J. Vis. Exp. (Pending Publication), e20983, doi: (2023).

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