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Related Experiment Video

Updated: Jun 21, 2025

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
09:38

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

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An efficient method for immortalizing mouse embryonic fibroblasts.

Srisathya Srinivasan1, Hsin-Yi Henry Ho1

  • 1Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, USA.

Biorxiv : the Preprint Server for Biology
|July 9, 2024
PubMed
Summary

This study presents an efficient CRISPR-based method to immortalize mouse embryonic fibroblasts (MEFs) by deleting the Tp53 gene. The resulting immortalized MEFs (iMEFs) are suitable for long-term genetic studies.

Keywords:
CRISPR/Cas9MEF immortalizationMEF isolationMouse embryonic fibroblasts (MEF)Tp53 knockoutimmortalized mouse embryonic fibroblasts (iMEFs)

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Area of Science:

  • Cell Biology
  • Genetics
  • Molecular Biology

Background:

  • Mouse embryonic fibroblasts (MEFs) are crucial for studying gene function and disease variants.
  • Primary MEFs senesce rapidly, limiting long-term genetic manipulation.
  • Existing immortalization methods are often inefficient or alter cell properties.

Purpose of the Study:

  • To develop an efficient and reliable protocol for immortalizing MEFs.
  • To overcome the limitations of primary MEF senescence for genetic studies.
  • To generate immortalized MEFs (iMEFs) that retain physiological characteristics.

Main Methods:

  • CRISPR-mediated deletion of the Tp53 gene in primary MEFs.
  • Optimized protocol for cell culture and immortalization.
  • Characterization of immortalized MEF properties.

Main Results:

  • Highly efficient generation of immortalized MEFs (iMEFs) within 14 days.
  • iMEFs closely resemble their parent primary MEF populations.
  • Individual iMEFs can be cloned and expanded for further research.

Conclusions:

  • The described CRISPR-based protocol provides an efficient method for MEF immortalization.
  • This method generates iMEFs suitable for long-term genetic manipulation and characterization.
  • The protocol has potential for immortalizing other mouse primary cell types.