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. 2015 Dec 8;27(8):2289–2308. doi: 10.1681/ASN.2015050565

Figure 4.

Figure 4.

Mice with podocyte-specific somatic ablation of GSK3β are healthy and have normal kidney function and histology. (A) Schematic diagram depicts the doxycycline-inducible tissue specific Cre-loxP gene recombination strategy to target the GSK3β gene selectively in glomerular podocytes in adult mice. By crossing the GSK3β-floxed mice with transgenic mice expressing rtTA under the control of podocin promoter and those expressing Cre driven by the tetracycline operator (tetO)-cytomegalovirus (CMV) promoter, the KO mice were bred. These mice carry double transgenes (Cre and rtTA transgenes) and homozygous floxed-GSK3β. In KO mice, rtTA transgene is specifically expressed in the glomerular podocytes. Feeding the KO mice with doxycycline (DOX), a compound enabling rtTA binding to the tetO promoter, will switch on Cre transgene expression in podocytes, which will subsequently execute the podocyte-specific excision of floxed exon 2 (E2) of GSK3β gene. (B) PCR genotyping of tail DNA from representative mice for detecting the Cre and rtTA transgenes and the floxed or WT GSK3β. All littermates lacking the Cre transgene were designated as control mice (Con). (C) Primary podocytes were prepared from glomeruli isolated from KO and control kidneys. The mRNA expression levels of GSK3α and GSK3β in primary podocytes were determined by real-time RT-PCR assay of the RNA samples extracted from the primary podocytes and presented as relative mRNA levels normalized to the endogenous GAPDH mRNA levels. The data are expressed as mean±SEM. *P<0.05 versus GSK3β expression levels in KO group (n=6). (D) Formalin-fixed, paraffin-embedded kidney specimens from KO mice or control littermates (Con) were sectioned and subjected to peroxidase immunohistochemistry staining for the β isoform of GSK3. Note that the staining of GSK3β was substantially diminished in the KO kidney, specifically in the periphery of glomerular tufts, in concert with the podocyte localization. As negative control, the primary antibody was replaced with preimmune IgG and no staining occurred. Arrowheads indicate podocytes positive for GSK3β in Con mice. Scale bar, 20 μm. (E and F) KO mice were phenotypically normal with serum creatinine levels (E) and kidney-to-body ratios (F) similar to those of control littermates at age 12 weeks. The data are expressed as mean±SEM. P>0.99 in E and P=0.70 in F (n=5).