Generation and characterization of an inducible renal proximal tubule-specific CreERT2 mouse

Figures

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  FIGURE 1. Generation of AMN CreERT2-mEGFP knock-in mice using the CRISPR/Cas9 strategy. (A) Schematic drawing depicting the wild-type allele of the mouse AMN gene, donor vector, and targeted allele. The donor construct, containing 1.5 kb 5′homologous arm (HL), 1.4 kb 3′homologous arm (HR), and the P2A-CreERT2-P2A-mEGFP gene cassette, was inserted into the last exon of the mouse AMN gene prior to the stop codon. E denotes exon. (B) Schematic diagram depicting the strategy for verifying on-target insertion using genomic PCR. The 5′set (purple) and 3′set (blue) of primers were used to confirm the precise insertion of the P2A-CreERT2-mEGFP-P2A cassette. (C) Representative agarose gel image of the PCR product from positive clones. The mutant allele for the 5′check was 3213 bp, while the mutant allele for the 3′check was 4124 bp. (D) Schematic representation depicting the strategy for confirming the mice with correct insertion using Southern blot. Xmn I (red arrow) and Stu I (yellow arrow) were utilized to digest the genomic DNA for Southern blot analysis. The 5′probe and EGFP probe were used to confirm accurate insertion of the P2A-CreERT2-mEGFP-P2A gene cassette. (E) Representative Southern…

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  FIGURE 2. Renal proximal tubular epithelial cell-specific expression and activation of the Cre recombinase in AMN CreERT2 mice. (A) Representative images showing the co-localization of GFP (green) and megalin (red) in renal proximal tubules. Rectangles depicted in the upper column were shown at a higher magnification in the lower column. (B) Diagram illustrating the generation of AMN CreERT2 ; Rosa26-LSL-tdTomato mice. (C) Expression of the tdTomato reporter gene in various tissues. The Cre activity (tdTomato+) was only detected in the small intestine and kidney of the AMN CreERT2 ; Rosa26-tdTomato mice following tamoxifen induction. (D–I) The Cre activity was detected in renal proximal tubules in the cortex and medulla, which was co-stained with Aqp1 and LTL (D–E). No Cre activity was detected in the thick ascending limb of the loop of Henle stained with NKCC2 (F), distal tubules stained with calbindin (G), collecting tubules stained with Aqp2 (H), and the glomerulus stained with podocalyxin (I). Scale bar = 100 μm (C, D) and 50 μm (A, E, F, G, H, I). The high dose of tamoxifen was used in this set of experiments.

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  FIGURE 3. Efficient recombination occurred in renal proximal tubular cells of AMN CreERT2 ; Rosa26-LTL-tdTomato mice after tamoxifen administration. (A) The induced Cre recombination efficiency in proximal tubule epithelial cells of AMN CreERT2 mice with different dosages of tamoxifen. In the control group, no leak Cre activity was observed in the whole kidney without tamoxifen induction. The total number of tdTomato-positive cells significantly increased with the rise in tamoxifen dosage. (B) Induction efficiency in different tamoxifen dose groups. The ratio of Aqp1+/tdTomato+ cells to Aqp1+ cells was measured in five randomly selected images per kidney (n = 4). Three asterisks signify highly significant differences between different groups. (**p < 0.001). (C) Cell specificity of CreERT2 activity in the renal proximal tubular epithelial cells. The ratio of Aqp1+/tdTomato+ cells to Aqp1-/tdTomato+ cells in five randomly selected images were measured per kidney (n = 4). Scale bar = 50 μm.

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  FIGURE 4. Knockout of the Exoc4 gene, specifically in renal proximal tubules in AMN CreERT2 ; Exoc4 fl/fl mice, led to albumin accumulation in the proximal tubular epithelial cells. (A) Diagram depicting the generation of tamoxifen-inducible AMN CreERT2/+ ; Exoc4 fl/fl mice. (B) Representative agarose gel image of RT-PCR products from renal cortices of vehicle- or tamoxifen-treated AMN CreERT2/+ ; Exoc4 fl/fl mice. The wild-type form of Exoc4 mRNA amplification product is 1602 bp, while the truncated form is 268 bp. (C) mRNA expression levels of Exoc4 in AMN CreERT2/+ ; Exoc4 fl/fl mice at day 14 after vehicle (corn oil) or tamoxifen administration using RT-qPCR (n = 3). An asterisk denotes a significant difference between two groups (*p < 0.05). (D) Immunohistochemistry staining of albumin accumulation in renal proximal tubules of AMN CreERT2/+ ; Exoc4 fl/fl mice at day 14 after vehicle or tamoxifen administration (n = 3). Rectangles depicted in the left column were shown at a higher magnification in the right column, respectively. The black arrowhead indicates albumin accumulation in the brush border of proximal tubular epithelial cells. PT denotes proximal tubule; DT denotes distal…