Tamoxifen-inducible podocyte-specific iCre recombinase transgenic mouse provides a simple approach for modulation of podocytes in vivo

Abstract

We report the generation and initial characterization of a mouse line expressing tamoxifen-inducible improved Cre (iCre) recombinase (iCre-ER(T2)) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin-iCreER(T2) mice. The efficiency of iCre activity was confirmed by crossing podocin-iCreER(T2) with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin-iCreER(T2);ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin-iCre-ER(T2) mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally-restricted manner.

FIG. 1

Schematic diagram of the podocin-iCreER^T2 construct. Human podocin promoter was inserted in the upstream of an iCre-ER^T2 expression cassette, which also contained an intron derived from the rabbit β-globin and a bovine growth hormone poly A (bGHpA) sequence. PCR primers, iCre-PCR-F (forward) and iCre-PCR-R (reverse), were used to determine the presence of the iCre transgene in mice.

FIG. 2

(a) Schematic diagram of the CMV-LoxP-Stop-LoxP-LacZ (CMV-LSL-LacZ) construct. (b) The podocin-iCre-ER^T2 construct was tested in podocytes in vitro by using CMV-LSL-LacZ, a floxed LacZ reporter. Podocytes were exposed to tamoxifen for 16 h and evaluated by β-gal staining. (I) control nontransfected podocytes, (II) transfected podocytes with podocin-iCre-ER^T2 construct, (III) podocytes transfected with both podocin-iCre-ER^T2 and LacZ floxed constructs but only exposed to vehicle for 16 h (IV) podocytes transfected with both constructs and exposed to tamoxifen (100 nM) for 16 h. β-gal activation is manifested by bright blue staining.

FIG. 3

iCre expression in kidneys of transgenic mice. (a) PCR analysis of genomic DNA isolated from the tail of 6-week-old podocin-iCreER^T2 transgenic mice crossed with R26R mice. Each lane represents DNA from a different mouse. The upper gel shows genotyping for podocin-iCre-ER^T2. The bottom gel exhibits genotyping for the presence of ROSA26 transgene. (b) RT-PCR for iCre in the kidneys treated with DNase I. PCR reactions were performed using templates from RT reactions with or without reverse transcriptase to control for DNA contamination (PCR control). Each lane represents DNA from a different mouse. Amplification of β-actin mRNA served as an additional control. (c) RT-PCR of iCre mRNA expression in extrarenal tissues from transgenic mice. Kidney tissue was used as control.

FIG. 4

Podocyte-specific LacZ activation in podocin-iCreER^T2; R26R double transgenic mice was assessed using X-gal staining. (a) podocin-iCreER^T2; R26R adult mice treated with vehicle (6–8 weeks old), (b) adult mice injected ip with tamoxifen (2 mg) for 5 days, (c) adult mice injected ip with tamoxifen (2 mg) for 10 days, and (d) adult mice injected ip with tamoxifen (4 mg) for 10 days. Kidneys were sectioned and stained for β-gal expression. Histological analysis for β-gal staining was evaluated five days after last ip injection. Several magnifications of the kidney sections with β-gal staining in the podocytes are shown with blue staining indicative of LacZ positive cells.

FIG. 5

(a) X-gal positive (blue) staining and nephrin positive (green) immunofluorescence staining were performed on the same cryosection. Nephrin positive cells corresponded to β-gal staining in Podocin-iCreER^T2, R26R double transgenic mice. (b) Immunostaining for nephrin (brown) merged with whole mount X-gal staining of adult kidneys (light blue). Arrows point to prominent cells (dark blue) that are positive for both β-gal and nephrin.