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 add PBS (Ca2+/Mg2+ free) to 10 mL. Count the cells.
- Pellet 106 cells and resuspend in 100 μl of Buffer R (included in the cell electroporation kit, see Table of Materials).
- Add plasmid DNA for transfection: 4.5 μg of transposable vector (epB-Bsd-TT-NIL or epB-Bsd-TT-NIP) and 0.5 μg of the piggyBac transposase plasmid.
- Transfect with the cell electroporation system (see Table of Materials) according to manufacturer’s instructions with the following parameters: 1,200 V voltage, 30 ms width, 1 pulse. Seed the cells in human iPSC medium supplemented with 10 μM Y-27632 (ROCK inhibitor, see Table of Materials) in a 6 mm matrix-coated dish.
Divulgaciones
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 |
Tags
Citar este artículo
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).