A novel ES cell line, TT2, with high germline-differentiating potency

Area of Science:

• Developmental Biology
• Genetics
• Stem Cell Biology

Background:

• Efficient germline colonization by gene-targeted or gene-trapped embryonic stem (ES) cells is crucial for producing mutant mice.
• Previous methods faced challenges in achieving consistent and high-level germline transmission.

Purpose of the Study:

• To establish and evaluate a new mouse embryonic stem (ES) cell line, TT2, for efficient germline transmission.
• To determine the optimal method for maximizing TT2 cell colonization and germline contribution in chimeric mice.

Main Methods:

• Established a new ES cell line (TT2) from an F1 hybrid embryo.
• Injected TT2 cells into blastocysts and eight-cell stage embryos.
• Assessed TT2 cell colonization in various tissues and germline contribution in resulting chimeric mice.
• Evaluated the germline-differentiating potency of TT2 cells over time and in independently isolated clones.

Main Results:

• Injection into eight-cell embryos, unlike blastocysts, led to efficient TT2 cell colonization in all tissues of chimeric pups.
• Chimeric pups were disproportionately male, with TT2-derived cells often dominating the cell population (>50% in >70% of males).
• Males derived from TT2 cells exclusively produced TT2-derived offspring, demonstrating stable germline transmission.
• Twenty-one of 24 mutant clones yielded germline chimeras, with 19 showing rates comparable to parental cells.

Conclusions:

• The TT2 ES cell line is a valuable tool for producing mutant mice with high germline transmission efficiency.
• Injecting ES cells into eight-cell embryos is a superior method for achieving widespread colonization and germline contribution.
• TT2 cells maintain stable germline-differentiating potency, making them reliable for generating genetically modified mouse models.