An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene
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, prima...
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Bio-protocol LLC
2025-01-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5159&type=0 |
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| author | Srisathya Srinivasan Hsin-Yi Ho |
| author_facet | Srisathya Srinivasan Hsin-Yi Ho |
| author_sort | Srisathya Srinivasan |
| collection | DOAJ |
| description | 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 inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. 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 broadly to immortalize other mouse primary cell types. |
| format | Article |
| id | doaj-art-563e2eee3d9d49d6a1b311d770656205 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-563e2eee3d9d49d6a1b311d7706562052025-02-07T08:16:38ZengBio-protocol LLCBio-Protocol2331-83252025-01-0115210.21769/BioProtoc.5159An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 GeneSrisathya Srinivasan0Hsin-Yi Ho1Department of Cell Biology and Human Anatomy, University of California, Davis, School of Medicine, Davis, CA, USADepartment of Cell Biology and Human Anatomy, University of California, Davis, School of Medicine, Davis, CA, USAMouse 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 inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. 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 broadly to immortalize other mouse primary cell types.https://bio-protocol.org/en/bpdetail?id=5159&type=0 |
| spellingShingle | Srisathya Srinivasan Hsin-Yi Ho An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene Bio-Protocol |
| title | An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene |
| title_full | An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene |
| title_fullStr | An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene |
| title_full_unstemmed | An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene |
| title_short | An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene |
| title_sort | efficient method for immortalizing mouse embryonic fibroblasts by crispr mediated deletion of the tp53 gene |
| url | https://bio-protocol.org/en/bpdetail?id=5159&type=0 |
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