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ER-based double iCre fusion protein allows partial recombination in forebrain
Emilio Casanova1, Sandra Fehsenfeld, Thomas Lemberger
1Deutsches Krebsforschungszentrum, Heidelberg, Germany. e.casanova@dkfz.de
Genesis (New York, N.Y. : 2000)
|October 24, 2002
Summary
A novel tamoxifen-inducible Cre fusion protein, ERiCreER, shows enhanced activity in cell culture but limited in vivo recombination in the brain. This suggests incomplete Cre-recombinase activation in neuronal cells despite tamoxifen binding.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Tamoxifen-inducible Cre recombinase systems are crucial for conditional gene manipulation in vivo.
- Previous Cre fusion proteins have shown varying efficacy and specificity.
- Targeting gene recombination to specific brain regions requires precisely controlled inducible systems.
Purpose of the Study:
- To generate and characterize a novel tamoxifen-inducible Cre fusion protein (ERiCreER) for brain-specific gene manipulation.
- To assess the activity and specificity of ERiCreER in cell culture and in vivo models.
- To evaluate the efficiency of ERiCreER-mediated recombination in the mouse brain.
Main Methods:
- Construction of a tamoxifen-inducible double Cre fusion protein (ERiCreER) by fusing ERT2 domains to iCre recombinase.
- Generation of a transgenic mouse line expressing ERiCreER in the brain under the CamKIIalpha promoter.
- Testing ERiCreER activity in vitro using cell culture assays.
- In vivo assessment of ERiCreER-mediated recombination in a CREB floxed mouse line following tamoxifen administration.
- Analysis of recombination by CREB allele status and CREM protein upregulation.
Main Results:
- ERiCreER demonstrated twofold increased activity in cell culture compared to MerCreMer.
- ERiCreER was successfully targeted to various brain regions including hippocampus, cortex, and hypothalamus.
- No background recombination was observed in vivo in the absence of tamoxifen.
- Tamoxifen administration led to nuclear translocation of ERiCreER but resulted in only 5-10% CREB allele recombination.
- CREM protein upregulation correlated with CREB recombination, confirming cellular-level activity.
Conclusions:
- ERiCreER is a functional tamoxifen-inducible Cre fusion protein with enhanced in vitro activity and brain-specific expression.
- Despite tamoxifen binding and nuclear translocation, ERiCreER exhibits limited in vivo recombination efficiency in the mouse brain.
- Further optimization may be required to achieve full Cre-recombinase activation in neuronal cells for effective gene editing.