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Published on: April 20, 2018
Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation
Johannes Hirrlinger1, Robert P Requardt, Ulrike Winkler
1N05 Neural Plasticity, Interdisciplinary Centre for Clinical Research (IZKF), University of Leipzig, Leipzig, Germany. johannes.hirrlinger@medizin.uni-leipzig.de
Plos One
|December 18, 2009
Summary
The novel split-CreERT2 system enables precise spatial and temporal control of DNA recombination. This advanced tool allows researchers to target specific cell populations in vivo using tamoxifen, enhancing genetic manipulation capabilities.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Conditional gene regulation using DNA recombination, like the Cre/LoxP system, is vital in biological research.
- Achieving precise spatiotemporal targeting of cells for recombination has been a significant challenge.
- Existing methods rely solely on promoter activity for spatial specificity, limiting temporal control.
Purpose of the Study:
- To develop a system for precise temporal control of split-Cre-mediated DNA recombination.
- To extend the split-Cre system by incorporating a tamoxifen-inducible domain (ERT2).
- To create a tool for spatiotemporal control of genetic recombination in vivo.
Main Methods:
- Fusion of split-Cre proteins with the tamoxifen-inducible ERT2 domain.
- Utilizing two independent Cre-dependent reporters in cultured cells.
- Testing various split-CreERT2 constructs for recombination efficiency and tamoxifen sensitivity.
Main Results:
- The NCre-ERT2+ERT2-CCre combination demonstrated favorable properties for the split-CreERT2 system.
- An induction ratio of approximately 10 was observed.
- Effective concentrations (EC50) for 4-hydroxy-tamoxifen ranged from 10 nM to 70 nM in vitro.
Conclusions:
- The split-CreERT2 system shows strong potential for inducing DNA recombination in mice with LoxP-flanked alleles.
- This system offers a new method for precise spatial and temporal genetic manipulation.
- It enables targeted genetic access to cell populations based on dual promoter activity and tamoxifen induction.
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