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Isolation and Handling of Mouse Embryonic Fibroblasts

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Mouse Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1194))

Abstract

Primary mouse embryonic fibroblasts (MEFs) are the most commonly used feeder layers that help to support growth and maintain pluripotency of embryonic stem cells (ESC) in long-term culture. Feeders provide substrates/nutrients that are essential to maintain pluripotency and prevent spontaneous differentiation of ESC. Since embryonic fibroblasts stop dividing after a few passages, care must be taken to isolate them freshly. Here, we provide a protocol to derive MEFs and describe the method to inactivate the cells using mitomycin C treatment. The protocol also describes freezing, thawing, and passaging of MEFs. This basic protocol works well in our laboratory. However, it can be modified and adapted according to any user’s particular requirement.

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Acknowledgement

This work was supported by Victoria Government’s Infrastructure Operational Program.

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Authors and Affiliations

  1. Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering, Monash University, Clayton, VIC, 3800, Australia

    Kanika Jain, Paul J. Verma & Jun Liu

  2. Turretfield Research Centre, South Australian Research & Development Institute, Holland Road, Rosedale, SA, 5350, Australia

    Paul J. Verma

Authors
  1. Kanika Jain
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  2. Paul J. Verma
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  3. Jun Liu
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Corresponding author

Correspondence to Paul J. Verma .

Editor information

Editors and Affiliations

  1. Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory,, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Shree Ram Singh

  2. Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

    Vincenzo Coppola

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Jain, K., Verma, P.J., Liu, J. (2014). Isolation and Handling of Mouse Embryonic Fibroblasts. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1215-5_13

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  • DOI: https://doi.org/10.1007/978-1-4939-1215-5_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1214-8

  • Online ISBN: 978-1-4939-1215-5

  • eBook Packages: Springer Protocols

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