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Updated: Jun 2, 2026
Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain
Published on: December 22, 2014
Development and characterization of transgenic mouse models for conditional gene knockout in the blood-brain and
Matthew H Crouthamel1, Edward J Kelly, Rodney J Y Ho
1Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195-7610, USA.
Abstract:
For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow conditional gene knockout using Cre/loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region (LPVcr) and to brain endothelium (the blood-brain-barrier) using the proximal promoter region of the human von Willebrand Factor gene (hVWF-f). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n-LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.
Insights
Researchers developed new transgenic mice to study blood-CNS barriers. These mice enable conditional gene knockout in the choroid plexus and brain endothelium, crucial for understanding drug delivery to the central nervous system (CNS).
Area of Science:
- Neuroscience
- Genetics
- Pharmacology
Background:
- Drug penetration into the central nervous system (CNS) is often restricted by the blood-CNS barriers.
- Understanding the role of protein components in these barriers is crucial for developing effective CNS-acting drugs.
- Conditional gene knockout technologies are essential for dissecting the function of specific genes within complex biological systems.
Purpose of the Study:
- To create transgenic mouse models for conditional gene knockout in specific cell types of the blood-CNS barriers.
- To enable the quantitative assessment of protein component roles in blood-CNS barrier function.
- To facilitate research on drug delivery and therapeutic strategies targeting the central nervous system.
Main Methods:
- Development of transgenic mice utilizing Cre/loxP technology for conditional gene knockout.
- Targeted expression of Cre-recombinase to the choroid plexus using the LPVcr and to brain endothelium using the hVWF promoter.
- Verification of cell-type specific Cre activity using the Z/EG dual reporter gene and analysis of EGFP expression via RT-PCR and immunohistochemistry.
Main Results:
- Successfully generated two transgenic mouse lines: LPV-Cre.0607 specifically expressing Cre in the choroid plexus, and hVWF-Cre.1304 expressing Cre in brain endothelium.
- Confirmed cell-type specific Cre-mediated endonuclease activity in the target cells without ectopic expression.
- Demonstrated the utility of these models for precise genetic manipulation within the blood-CNS barrier components.
Conclusions:
- The developed transgenic mouse lines provide powerful tools for investigating the molecular mechanisms of the blood-CNS barriers.
- These models will aid in quantifying the contribution of specific proteins to barrier function and drug permeability.
- This research paves the way for improved strategies in CNS drug development and delivery.
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