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Popping Out MX Cassettes from Saccharomyces cerevisiae
1Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina 27710 john.mccusker@duke.edu.
Cold Spring Harbor Protocols
|April 5, 2017
Summary
MX cassettes enable efficient gene knockout (KO) mutations in yeast. Recycling these cassettes via recombination or site-specific methods allows for reuse and scar-free mutations, streamlining genetic engineering.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- MX cassettes are widely used for generating knockout (KO) mutations in Saccharomyces cerevisiae.
- Recycling MX cassettes by
- popping out
Purpose of the Study:
- To describe methods for recycling MX cassettes in yeast.
- To highlight the utility of counterselections for genetic manipulation.
- To explain the generation of scar-free mutations.
Main Methods:
- Spontaneous recombination between MX3 or PR direct repeats.
- Cre-mediated recombination between loxP direct repeats.
- Utilizing counterselectable markers (URA3MX, LYS5MX, FCA1/FCY1MX, amdSYM).
Main Results:
- MX cassette recycling allows for reuse in generating additional KO mutations.
- Popping out MX cassettes removes homology, facilitating further KO generation.
- Counterselections enable positive selection for various genetic manipulations, including cassette recycling.
- Seamless or
- scar-free
Conclusions:
- MX cassette recycling is a valuable technique for efficient yeast genetic engineering.
- Counterselections significantly enhance the versatility of MX cassettes.
- Seamless mutations can be generated by popping out integrated counterselectable MX cassettes.