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Updated: Jun 21, 2025
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Published on: June 21, 2012
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.
Abstract:
Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inefficient or alter the physiological properties of the cells. Here, we describe an optimized protocol for immortalizing MEFs via CRISPR-mediated deletion of the Tp53 gene. This method is highly efficient and consistently generates immortalized MEFs, or iMEFs, within 14 days. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted to immortalize other mouse primary cell types.
Insights
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.
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.
