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Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression
Margaret L Van Keuren1, Galina B Gavrilina, Wanda E Filipiak
1Transgenic Animal Model Core, University of Michigan Medical School, 2560 MSRB II, Ann Arbor, MI 48109, USA.
Transgenic Research
|April 28, 2009
Summary
Generating transgenic mice using bacterial artificial chromosomes (BACs) requires specific DNA purification, polyamine buffer, and genotyping for consistent results. BAC transgenesis is a valuable tool for studying gene expression and disease models.
Area of Science:
- Biotechnology
- Genetics
- Molecular Biology
Background:
- Transgenic mice are crucial models in biomedical research for studying gene function and disease.
- Bacterial artificial chromosomes (BACs) enable physiological gene expression in transgenic models, mimicking endogenous gene patterns.
Purpose of the Study:
- To optimize the generation of transgenic mice using BAC transgenes.
- To identify critical factors influencing BAC transgenesis efficiency and success rates.
Main Methods:
- Generated 707 transgenic founders from 86 BAC transgenes using three DNA purification methods.
- Investigated the impact of BAC DNA purification, polyamine microinjection buffer, BAC size, and DNA concentration on transgenesis outcomes.
- Utilized sensitive genotyping assays to confirm BAC integration and expression.
Main Results:
- Transgenesis efficiency was consistent across different BAC DNA purification methods.
- Polyamine microinjection buffer was essential for intact BAC transgene integration.
- Optimal transgenic efficiency was achieved within a narrow DNA concentration range; high concentrations reduced birth rates.
- Complete BAC integrations were confirmed in 47 BACs, with fragment integrations in 65%.
- Gene expression was observed in 80% of founders from tested BAC transgenes.
Conclusions:
- Consistent and reproducible BAC transgenesis relies on optimized DNA purification, polyamine buffer use, and sensitive genotyping.
- BAC transgenesis is a reliable method for creating transgenic mouse models with predictable gene expression.
- This study provides a framework for improving the efficiency and success of BAC-mediated transgenesis.
