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A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9
Yang Wang1,2,3, Shuaitao Hu1,2,3,4, Chunsheng Han1,2,3,4
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Current Protocols
|September 12, 2024
Summary
This study presents streamlined protocols for establishing mouse germline stem cell (GSC) lines and performing gene knock-in using CRISPR/Cas9. These efficient methods enable the generation of genetically modified GSCs within three months for research applications.
Area of Science:
- Stem Cell Biology
- Reproductive Biology
- Molecular Genetics
Background:
- Mammalian spermatogonial stem cells (SSCs), or germline stem cells (GSCs), are crucial for fertility preservation, gene therapy, and animal breeding.
- Establishing and genetically modifying mouse GSCs (mGSCs) in vitro presents challenges due to slower proliferation and transfection resistance compared to pluripotent stem cells.
- Ongoing advancements aim to simplify and enhance mGSC culture and gene editing techniques.
Purpose of the Study:
- To describe streamlined protocols for developing mGSC lines from small mouse testis samples.
- To detail a method for gene knock-in in mGSCs utilizing CRISPR/Cas9 technology.
- To provide researchers with efficient procedures for generating genetically modified GSCs.
Main Methods:
- Establishment of mGSC lines from small testicular samples.
- Preparation of plasmids for gene knock-in via CRISPR/Cas9.
- Gene knock-in in mGSCs using electroporation for gene delivery.
Main Results:
- A streamlined procedure combining three basic protocols was detailed.
- Site-specific knock-in mGSC lines can be obtained within a 3-month timeframe.
- The protocols are designed to be simple and efficient for researchers.
Conclusions:
- The described protocols facilitate the development of gene-modified GSCs.
- These methods can aid researchers in exploring scientific questions using GSCs.
- The protocols may inform GSC research in other mammalian species.
