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Protocol for large DNA transgenesis in mice using the Cas9+Bxb1 toolbox
Valerie Erhardt1, Chrystal M Snow1, Vishnu Hosur1
1The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME, USA.
STAR Protocols
|April 16, 2024
Summary
This study introduces a new protocol for large DNA transgenesis in mice using the Cas9+Bxb1 toolbox. This method enables precise integration of large DNA constructs into mouse zygotes, aiding cancer research.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Transgenic Animal Models
Background:
- Precise integration of large DNA constructs (>3 kb) into mouse zygotes is a significant challenge in genetic engineering.
- Existing methods often lack efficiency and accuracy for large DNA insertions.
Purpose of the Study:
- To present a robust protocol for large DNA transgenesis in mice.
- To enable efficient and precise integration of large DNA constructs using a novel toolbox.
Main Methods:
- Utilized the Cas9+Bxb1 toolbox for targeted DNA integration.
- Developed a protocol involving selection of specific mouse strains with preplaced attachment sites.
- Employed microinjection of plasmid donor DNA into mouse zygotes followed by recombination-mediated cassette exchange.
Main Results:
- Successfully demonstrated a protocol for large DNA transgenesis in mice.
- The method facilitates precise integration of DNA constructs exceeding 3 kb.
Conclusions:
- The developed Cas9+Bxb1 based protocol offers a powerful approach for generating transgenic mice with large DNA insertions.
- This technique has potential applications in studying the functional impact of large structural variations, particularly in cancer research.
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