Fig 1.
Generation of Pax8-CreERT2 transgenic mice.
(A) The previously published pPax8-rtTA plasmid [7] is shown in the top panel. The CreERT2 coding sequence followed by a polyadenylation signal (pA) was used to replace the rtTA sequence of pPax8-rtTA and generate plasmid pPax8-CreERT2 (bottom panel) containing 4.3kb of upstream regulatory sequence, complete exon 1 and intron 1, part of exon 2 and 0.8kb of intron 2 of the murine Pax8 gene. (B) FISH analysis of metaphase spreads using pPax8-CreERT2 as probe (green) maps the integration site to chromosome 6 (arrowheads). A weaker signal from the endogenous Pax8 locus can be detected on chromosome 2 (arrows).
Fig 2.
Pax8-CreERT2 mediated β-galactosidase expression.
(A) Enzymatic X-gal staining of cryosections of kidneys (top panels) derived from 10 week old Pax8-CreERT2/Rosa26R mice either induced with tamoxifen (a, renal parenchyma; b, collecting ducts) or uninduced (c). Tamoxifen administration did not induce β-galactosidase expression in any of the other major organs examined (representative tissues presented in bottom panels). (B) β-galactosidase positivity was exclusively found in the tubular epithelium of the kidney of tamoxifen treated mice, with no staining observed in glomeruli or bloods vessels (a, framed area is shown enlarged in b). Kidneys from vehicle treated mice were negative (c). Co-localisation studies revealed X-gal expression within tubules of all renal tubular compartment (proximal tubules—AQP1, distal tubules–THP and collecting ducts–AQP2). Scale bars, 100μm.
Fig 3.
Generation of Slc22a6-CreERT2 knock-in mice.
(A) Schematic diagram of the generation of Slc22a6-CreERT2 knock-in mice. The exon-intron structure of the mouse Slc22a6 locus is shown at the top. The targeting vector has a 4.3 kb 5′ arm that locates immediately upstream of the Slc22a6 gene ATG, the CreERT2-pA coding sequence, a PGK-hygromycin selection cassette flanked by FRT sites (green triangles) and a 5.3 kb 3′ arm that extends to intron 6. (B) Southern blots show a control (wild-type) and 4 correctly targeted ES cell clones (77, 79, 92, 185) that give expected hybridisation patterns. Clone 142 is incorrectly targeted. The location of 5’, 3’ and internal probes as well as expected band lengths are indicated in the diagrams on the right.
Fig 4.
Slc22a6-CreERT2 mediated β-galactosidase expression.
(A) Enzymatic X-gal staining of cryosections of kidneys (top panels) derived from 10 week old Slc22a6-CreERT2/Rosa26R mice either induced with tamoxifen (a, renal cortex; b, medulla) or uninduced (c, renal cortex). Tamoxifen administration did not induce β-galactosidase expression in any of the other major organs examined (representative tissues presented in bottom panels). (B) β-galactosidase positivity was exclusively found in the tubular epithelium of the kidney of tamoxifen treated mice (a); kidneys from vehicle treated mice were negative (b). Co-localisation studies revealed specific X-gal expression within proximal tubular epithelium (AQP1) with no expression detected in distal tubules (THP) or collecting ducts (AQP2). Scale bars, 100μm.
Fig 5.
Renal tubule specific models of Vhl deletion.
(A) PCR analysis of recombination at the Vhl locus in the kidneys of mice with combinations of Pax8-CreERT2, Slc22a6-CreERT2 and the Vhl floxed (fl) and wild-type (+) alleles. The positions of the bands representing the Vhl floxed, wild type (Wt) and recombined (Δ) alleles are indicated. (B) Histological images of representative renal sections from 12 month old control, Pax8-CreERT2/VhlΔ/Δ and Slc22a6-CreERT2/VhlΔ/Δ mice (stains and antibodies as indicated, arrowheads indicate abnormal vascularisation). Scale bars, 100μm.