Inducible site-directed recombination in mouse embryonic stem cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Cre recombinase enzyme is a powerful tool for site-directed DNA recombination, enabling gene deletion or expression.
• Controlling the activity of Cre recombinase remotely is highly desirable for precise genetic manipulation in biological systems.
• Existing methods for Cre-mediated recombination lack temporal or spatial control, limiting their application.

Purpose of the Study:

• To develop a tamoxifen-inducible Cre recombinase system for remote control of gene activity.
• To create fusion proteins linking Cre recombinase with a mutated estrogen receptor hormone-binding domain.
• To demonstrate the efficacy of tamoxifen in inducing Cre-mediated recombination in embryonic stem cells.

Main Methods:

• Construction of expression vectors encoding Cre recombinase fused to a mutated murine estrogen receptor hormone-binding domain.
• Utilizing embryonic stem cells expressing these fusion proteins.
• Treatment with tamoxifen or 4-hydroxy-tamoxifen to induce Cre-mediated recombination.
• Monitoring recombination via activation of a stably integrated LacZ reporter gene.

Main Results:

• Tamoxifen efficiently induces Cre-mediated recombination in embryonic stem cells.
• Recombination of the LacZ gene is complete within 3-4 days upon tamoxifen administration (10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen).
• Fusion proteins with tamoxifen-binding domains on both ends offer even tighter control over Cre enzymatic activity.

Conclusions:

• A tamoxifen-inducible Cre recombinase system has been successfully developed and validated in embryonic stem cells.
• This system provides a new and valuable genetic tool for temporal and spatial control of gene mutation or deletion.
• Transgenic mice expressing this inducible Cre enzyme hold significant potential for developmental biology and adult animal studies.