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Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells
Published on: October 5, 2011
Transfecting and nucleofecting human induced pluripotent stem cells
Papri Chatterjee1, Yuri Cheung, Chee Liew
1UCR Stem Cell Center, Department of Cell Biology and Neuroscience, University of California Riverside, USA.
Journal of Visualized Experiments : Jove
|October 19, 2011
Summary
This study presents a robust method for genetically modifying human induced pluripotent stem cells (hiPSCs) using nucleofection. The protocol ensures efficient gene delivery and stable integration without affecting the cells
Area of Science:
- Stem Cell Biology
- Genetic Engineering
- Molecular Biology
Background:
- Genetic modification is crucial for stem cell research and potential clinical applications.
- Transfection of human embryonic stem cells (HESCs) is challenging, and methods for human induced pluripotent stem cells (hiPSCs) are not well-established.
Purpose of the Study:
- To demonstrate a reliable protocol for the transfection and nucleofection of human iPSCs.
- To establish a method for generating stable, transgene-expressing hiPSC lines.
Main Methods:
- Human iPSCs were cultured feeder-free to prevent feeder cell transfection.
- Nucleofection was performed using a plasmid with an enhanced green fluorescence protein (eGFP) reporter.
- Cells were pre-treated with ROCK inhibitor, trypsinized, nucleofected, and replated on feeders in conditioned medium.
Main Results:
- Transgene expression in human iPSCs was observed as early as 6 hours post-nucleofection.
- Stable transgenic hiPSC lines were obtained within 1 week using antibiotic selection.
- The protocol is robust, reproducible, and maintains the pluripotency of hiPSCs.
Conclusions:
- A reliable and efficient nucleofection protocol for human iPSCs has been established.
- This method facilitates the genetic modification of hiPSCs for research and therapeutic development.
- The protocol ensures the maintenance of hiPSC pluripotency throughout the process.
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