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Updated: Aug 29, 2026
Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain
Published on: May 20, 2022
Labeling of three different mouse ES cell lines with the green fluorescent protein
Wen-Ning Zhao1, Guo-Liang Meng, You-Fang Xue
1College of Life Science, Peking University, Beijing 100871, China. zhouym@pku.edu.cn
Abstract:
The linearized plasmid pEGFP-N3 was electroporated into three different mouse ES cell lines MESPU-13, MESPU-35 and MESPU-62 derived from 129/ter, C57BL/6J and BALB/c mouse strains respectively. Resistant clones were selected in the presence of G418 and then were identified under the fluorescence microscope through blue exciting light. Positive green clones were primarily expanded and further sorted using FACS(fluorescence activated cell sorter). Finally five EGFP stable integrated cell strains were obtained and were expanded (2 strains from 129/ter, 1 strain from C57BL/6J and 2 strains from BALB/c). Each of the five cell strains presents high proliferation growth rate and typical morphology characters of ES cells and their colonies. More than 85% cells of each cell strain contain normal diploid karyotype. Then some analysis such as the AP (alkaline phosphatase) staining, oct4 gene expression assay, embryonic body formation and differentiated test in vivo and in vitro were made. The results indicated that the stable labeled ES cell strains had the normal karyotypes and maintained the ES cell typical characteristics.
Insights
Researchers created stable, fluorescent mouse embryonic stem cell (ES cell) lines using electroporation. These new EGFP-expressing ES cell lines maintain normal karyotypes and key stem cell characteristics for research applications.
Area of Science:
- Stem Cell Biology
- Molecular Biology
- Genetics
Background:
- Mouse embryonic stem cells (ES cells) are crucial for developmental biology and regenerative medicine research.
- Establishing stable, genetically modified ES cell lines is essential for studying gene function and developing cell-based therapies.
- Previous methods for genetic modification and selection of ES cells can be time-consuming and may affect cell viability.
Purpose of the Study:
- To generate stable, enhanced green fluorescent protein (EGFP)-expressing mouse ES cell lines from different mouse strains.
- To characterize the obtained cell lines for pluripotency, genetic stability, and differentiation potential.
- To provide reliable tools for future research in developmental biology and stem cell applications.
Main Methods:
- Linearized pEGFP-N3 plasmid electroporation into three mouse ES cell lines (MESPU-13, MESPU-35, MESPU-62).
- Selection of resistant clones using G418 and identification via fluorescence microscopy.
- Expansion and sorting of positive clones using fluorescence-activated cell sorting (FACS).
- Karyotype analysis, alkaline phosphatase (AP) staining, Oct4 gene expression assay, and in vitro/in vivo differentiation tests.
Main Results:
- Five stable EGFP-expressing ES cell strains were successfully generated from 129/ter, C57BL/6J, and BALB/c mouse strains.
- All five cell strains exhibited high proliferation rates, typical ES cell morphology, and normal diploid karyotypes (>85%).
- The stable labeled ES cell lines retained key stem cell characteristics, including AP staining, Oct4 expression, and differentiation potential.
Conclusions:
- Stable EGFP-labeled mouse ES cell lines with maintained pluripotency and genetic stability were successfully established.
- These cell lines serve as valuable tools for tracking and analyzing ES cell behavior in various biological contexts.
- The developed method provides a robust approach for generating genetically modified, functional ES cell lines for research purposes.
