A scalable embryonic stem cell-based platform for efficient generation of mitochondrial DNA mutant mice

Area of Science:

• Mitochondrial biology
• Genetics
• Animal models

Background:

• Mitochondria are crucial for cellular energy and signaling.
• Mitochondrial DNA (mtDNA) mutations are linked to human diseases.
• Existing animal models for mtDNA variation are limited.

Purpose of the Study:

• To develop a scalable platform for generating mice with diverse mtDNA mutations.
• To create a resource for studying the impact of mtDNA variation on physiology and disease.
• To investigate the role of mitochondrial function in early embryonic development.

Main Methods:

• Utilized an error-prone mtDNA polymerase for random mutagenesis.
• Employed a multiplexed cybrid fusion strategy to transfer mtDNA mutations into embryonic stem (ES) cells.
• Developed optimized ES cell-embryo aggregation for producing chimeric mice with germline transmission.
• Generated a library of ES cell lines with various homoplasmic and heteroplasmic mtDNA mutations.

Main Results:

• Successfully generated 155 donor fibroblast lines with distinct mtDNA mutations.
• Created 34 female ES cell lines with 18 different mtDNA mutations at various heteroplasmy levels.
• Produced chimeric mice with germline transmission of mtDNA mutations.
• Observed diverse mitochondrial phenotypes, including impaired oxidative phosphorylation and altered reactive oxygen species levels.
• Demonstrated a correlation between mitochondrial function and early embryonic development, indicating an energetic threshold.

Conclusions:

• The developed platform enables efficient production of mtDNA mutant mice.
• This resource facilitates systematic investigation of mtDNA variation in health, disease, and adaptation.
• Findings suggest a critical role for mitochondrial energy production in embryonic development.