[Targeting of MT-II gene in mouse ES cells]

Area of Science:

• Gene targeting and molecular biology
• Mammalian cell line engineering
• Developmental biology

Context:

• Developing genetically modified cell lines is crucial for studying gene function and disease.
• Mouse embryonic stem cells (mESCs) are a key model system for developmental and genetic studies.
• Efficient gene targeting methods are needed to create precise genetic modifications in stem cells.

Purpose:

• To generate homologous recombinant mouse embryonic stem cells (mESCs) for the metallothionein-II (MT-II) gene.
• To establish and characterize genetically modified mESC lines with pluripotency.
• To demonstrate the potential for generating chimeric animals from these engineered cells.

Summary:

• A gene targeting vector, pMT-II6.7, containing 6.7kb sequences homologous to the mouse metallothionein-II (mMT-II) gene and its flanking regions, was introduced into Mespu22 cells via electroporation.
• Out of 104 G418 and Ganc resistant clones, 26 positive clones were identified using PCR. Karyotype analysis revealed two clones (5-2 and 8-4) with high percentages (84% and 88%) of normal karyotypes.
• Southern blot analysis confirmed these two clones as homologous recombinants. Both cell lines maintained their pluripotency, capable of differentiation in vitro and in vivo. A chimeric mouse was successfully generated using cells from clone 8-4.

Impact:

• Successfully generated homologous recombinant mESC lines with high karyotypic stability.
• Demonstrated the retention of pluripotency in engineered mESC lines, enabling further developmental studies.
• Established a foundation for using these genetically modified cells in creating disease models and studying gene function in vivo, as evidenced by the chimeric mouse generation.