title: Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.
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-typemice 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-typeand hNphs2.Cre mice to Phb2fl/flmice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-typemice 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.
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Sybille
authors.initials: S
authors.lastname: Koehler
authors.affiliation: 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.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brähler
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Braun
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Hagmann
authors.affiliation: 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.
authors.forename: Markus M
authors.initials: MM
authors.lastname: Rinschen
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Späth
authors.affiliation: 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.
authors.forename: Martin
authors.initials: M
authors.lastname: Höhne
authors.affiliation: Max Planck Institute for Metabolism Research, Cologne, Germany.
authors.forename: F Thomas
authors.initials: FT
authors.lastname: Wunderlich
authors.affiliation: 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.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schermer
authors.affiliation: 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.
authors.forename: Thomas
authors.initials: T
authors.lastname: Benzing
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Electronic address: paul.brinkkoetter@uk-koeln.de.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Brinkkoetter
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D003062:Codon; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D019426:Integrases / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010455:Peptides / Q000235:genetics*; D050199:Podocytes / Q000378:metabolism*; D000091402:Prohibitins; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics*; D000097573:Red Fluorescent Protein
chemical_list: D003062:Codon; D019426:Integrases; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010455:Peptides; D000091402:Prohibitins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C407223:NPHS2 protein; C000717333:Phb2 protein, mouse; C045073:Cre recombinase
keywords: Cre recombinase; glomerulus; podocyte