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Kidney Int. 2017 Jun;91(6):1510-1517. doi: 10.1016/j.kint.2016.12.011. Epub 2017 Feb 7.

Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

Author information

1
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
2
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department of Pathology and Immunology, Division of Immunobiology, Washington University School of Medicine, St Louis, Missouri, USA.
3
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
4
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany.
5
Max Planck Institute for Metabolism Research, Cologne, Germany.
6
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Electronic address: paul.brinkkoetter@uk-koeln.de.

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.

KEYWORDS:

Cre recombinase; glomerulus; podocyte

PMID:
28187984
DOI:
10.1016/j.kint.2016.12.011
[Indexed for MEDLINE]

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