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Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination
Published on: August 1, 2011
Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent
Sybille Koehler1, Sebastian Brähler2, Fabian Braun1
1Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Abstract:
Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.
Insights
Researchers developed a new mouse model for studying kidney podocyte health. This tricistronic model offers improved gene editing efficiency and fluorescent labeling for better podocyte research and disease modeling.
Area of Science:
- Nephrology
- Genetics
- Molecular Biology
Background:
- Podocyte injury is central to glomerular diseases, driving proteinuria and kidney scarring.
- Understanding podocyte molecular mechanisms is crucial for therapeutic development.
- Existing mouse models like hNphs2.Cre facilitate podocyte gene manipulation but have limitations.
Purpose of the Study:
- To create an improved podocyte-specific mouse model for genetic manipulation and lineage tracing.
- To combine enhanced Cre recombinase activity with fluorescent labeling in a single, efficient tool.
- To validate the model's safety, efficacy, and utility in disease contexts.
Main Methods:
- Designed a novel tricistronic mouse model (Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type) using the Nphs2 promoter and viral T2A-peptides.
- Confirmed independent expression of podocin, Cre, and mTomato via immunofluorescence and flow cytometry.
- Assessed Cre recombination efficiency and disease phenotype in comparison to conventional hNphs2.Cre mice using Phb2 knockout models.
Main Results:
- The novel tricistronic model demonstrated normal development without glomerular pathology or off-target effects.
- Achieved superior gene recombination efficiency compared to conventional hNphs2.Cre mice.
- Podocyte-specific Phb2 knockout using the new model exacerbated glomerular injury more than with the hNphs2.Cre model.
Conclusions:
- The Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mouse is the first tricistronic model for enhanced podocyte-specific gene editing and fluorescent labeling.
- This model offers improved Cre efficiency and simplifies research by eliminating the need for separate reporter lines.
- It provides a powerful new tool for investigating podocyte function and glomerular disease pathogenesis.
