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Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors
Munehiro Uemura1, Youko Niwa, Naoki Kakazu
1Evolutionary Medicine, Shiga Medical Center Research Institute, Moriyama, Japan.
Plos One
|March 31, 2010
Summary
Researchers developed a novel Cre-mediated chromosomal recombination system to simplify gene manipulation in mammalian cells. This technique facilitates the study of chromosomal context and disease modeling for translocations and copy-number variations.
Area of Science:
- Genomics
- Molecular Biology
- Cell Biology
Background:
- Chromosomal manipulation in mammalian cells, while valuable for gene regulation studies, faces significant technical challenges limiting its widespread adoption.
- Existing methods often involve complex procedures and leave substantial artificial elements at rearrangement junctions.
Purpose of the Study:
- To develop an improved Cre-mediated chromosomal recombination system to overcome technical hurdles in mammalian cell gene manipulation.
- To enable precise understanding of gene regulation within the chromosomal context and facilitate disease modeling.
Main Methods:
- Developed a Cre-mediated recombination system utilizing fluorescent proteins and site-specific recombinases for efficient targeting vector construction.
- Applied the system to a human cell line to recapitulate pathogenic chromosomal translocations (MYC/IgH, BCR/ABL1).
- Induced intrachromosomal recombination between loxP sites to mark deletions or duplications with distinct fluorescent signals.
Main Results:
- Successfully recapitulated two major pathogenic chromosomal translocations (MYC/IgH and BCR/ABL1) in a human cell line.
- Demonstrated that intrachromosomal recombination frequency is inversely proportional to the distance between recombination sites.
- Enabled easy identification of chromosome-rearranged cells with minimal artificial elements at junctions.
Conclusions:
- The developed Cre-mediated system simplifies chromosomal manipulation in mammalian cells, overcoming previous technical difficulties.
- This method facilitates the creation of cellular and animal models for diseases involving translocations and copy-number variations.
- The system's ability to use recombination frequency as a proximity sensor opens new avenues in genome biology research.
Related Concept Videos
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...
The recognition sites for Cre recombinase called LoxP...
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...