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Segmental genomic replacement in embryonic stem cells by double lox targeting
S Soukharev1, J L Miller, B Sauer
1Laboratory of Biochemistry and Metabolism, National Institute of Diabetes, Digestive and Kidney Disease, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Nucleic Acids Research
|September 3, 1999
Summary
This study introduces a novel Cre-mediated double lox recombination method for precise gene insertion in embryonic stem cells. This technique enables efficient and repeated knock-ins, improving transgene expression accuracy in mice.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- Precise genomic engineering in mice is crucial for studying gene function and disease.
- Current methods for gene knock-ins in embryonic stem (ES) cells can be inefficient and lack flexibility for repeated modifications.
Purpose of the Study:
- To develop a robust and efficient method for repeated gene knock-ins at a specific locus in ES cells.
- To facilitate accurate temporal and spatial transgene expression in mouse models.
Main Methods:
- Applied Cre-mediated double lox recombination in ES cells.
- Inserted a double lox cassette upstream of the MHL-1 gene using homologous recombination.
- Utilized heterospecific lox sites for targeted replacement with incoming DNA vectors.
Main Results:
- Achieved Cre-mediated replacement of genomic segments at a frequency three times higher than random DNA integration.
- Successfully identified correctly targeted ES colonies via PCR screening without drug selection.
- Demonstrated that recombination efficiency is independent of promoter activity at the target locus.
Conclusions:
- The described double lox recombination strategy is broadly applicable for targeted knock-ins in ES cells.
- This method offers a powerful tool for precise molecular engineering of the mouse genome.
- Facilitates improved accuracy in transgene expression for mouse models.