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Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation
Published on: November 17, 2017
Recombineering-based procedure for creating Cre/loxP conditional knockouts in the mouse.
1UNC-Chapel Hill, Chapel Hill, North Carolina, USA.
Current Protocols in Molecular Biology
|January 27, 2009
Summary
This study simplifies creating conditional knockout (CKO) mouse models using bacterial artificial chromosomes and recombineering. The streamlined method aids in generating CKO constructs for disease research and gene function studies.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Animal Models
Background:
- Gene targeting in mice is crucial for understanding gene function and modeling human diseases.
- Traditional methods for generating knockout models can be complex and time-consuming.
Purpose of the Study:
- To present a simplified method for preparing conditional knockout (CKO) constructs in mice.
- To facilitate the generation of CKO mouse models for research.
Main Methods:
- Utilizes bacterial artificial chromosomes, Cre/loxP and FLPe/FRT systems.
- Employs advanced recombineering techniques for construct preparation.
- Provides detailed guidelines for construct design and troubleshooting.
Main Results:
- Successfully generated over 30 CKO constructs.
- Demonstrated successful targeting of mouse embryonic stem cells with these constructs.
- Established mutant mouse lines from the generated CKO constructs.
Conclusions:
- The described method offers an efficient approach to generate CKO mouse models.
- This technique simplifies the creation of valuable tools for genetic research and disease modeling.
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02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.