Generation of Pax8-rtTA mice
We isolated a 5,636–base pair fragment from the mouse Pax8
gene that included 4.3 kilobases (kb) of the putative promoter along with complete exon 1, intron 1, exon 2 and part of intron 2. We modified the region around the translation initiation codon, amplified rtTA2s
-M2 (ref. 6
) by PCR and inserted it into the Pax8
promoter construct (Supplementary Fig. 1 online
After pronuclear injection of the 6.6-kb Pax8-rtTA insert DNA, we identified two transgenic founders among 72 offspring mice. Only one of those lines, designated Pax8-rtTA, transmitted the transgene and is described here.
Using multicolor fluorescence in situ
hybridization (M-FISH) and two-color FISH analysis, we mapped the site of integration of the Pax8
-rtTA construct to mouse chromosome 8 band B2 (Supplementary Fig. 2 online
). We then used ligation-mediated PCR similar to detection of integrated papillomavirus sequences (DIPS)-PCR assay7
to amplify the fusion region between integrated Pax8
-rtTA and adjacent genomic DNA (Supplementary Methods online
). Sequence analysis of 14 individual DIPS products allowed us to deduce that there were five to six copies of the Pax8
-rtTA construct integrated into a mouse L1 Line repetitive element (data not shown).
Functional Pax8-rtTA activity in vivo
To show functional expression of rtTA in vivo, we crossbred Pax8-rtTA mice with NZL-2 mice, a line with a tetracycline-responsive reporter encoding nuclear β-galactosidase (). Doxycycline was given to double-transgenic (Pax8-rtTA/NZL-2) mice at 12 weeks of age for 10 d. The kidneys of treated mice but not of control mice showed strong β-galactosidase expression in all renal tubular cells (). Within the liver, β-galactosidase expression was found in a small subset of hepatocytes that were located periportally. However, no expression was detected in the remaining hepatocytes or in any other tissue tested, including heart, lung, brain, spleen, thyroid and colon ().
Figure 1 Pax8-rtTA–mediated, doxycycline-controlled β-galactosidase expression in adult tissues. (a) The Pax8 promoter directs the expression of rtTA, which, in the presence of doxycycline (Dox), binds and transactivates the tetracycline-responsive (more ...)
Closer examination of the kidney revealed uniform β-galactosidase positivity in the renal cortex along all proximal and distal tubules and all collecting ducts (). Additionally, in the medulla, tubules and collecting ducts invariably stained positively (). In contrast, parietal and visceral epithelial cells of glomeruli, mesangial cells and blood vessels did not stain (). Only proximal tubular epithelial cells, when extending into the glomerular parietal epithelium, conferred β-galactosidase positivity to glomeruli ().
Figure 2 Pax8-rtTA–mediated, doxycycline-controlled β-galactosidase expression in adult renal tubular cells. (a) β-galactosidase positivity (nuclear staining) in Pax8-rtTA/NZL-2 double-transgenic mice induced with doxycycline was found (more ...)
Essentially the same pattern of activity was found in triple-transgenic Pax8
rtTA/LC-1/Rosa26R mice (), in which the expression of Cre recombinase from the LC-1 transgene8
is triggered and then activates the conditional cytosolic Rosa26 β-galactosidase reporter9
To investigate rtTA expression in mouse embryos, we mated Pax8-rtTA and NZL-2 mice under continuous treatment with doxycycline. Using whole-mount β-galactosidase staining of embryonic day 10.5 (E10.5) embryos, we found weak expression in the mid-hindbrain region and cloaca, whereas we detected strong expression in the caudal part of the hindbrain (). Cross-sections from the same embryos revealed additional Pax8-rtTA activity in mesonephric tubules, the Wolffian duct and the ureteric bud invading the metanephric mesenchyme ().
Figure 3 Pax8-rtTA–mediated, doxycycline-controlled β-galactosidase expression in embryonic kidney. (a) Whole-mount embryo (E10.5) stained for β-galactosidase activity. β-galactosidase–positive cells were macroscopically (more ...)
Induction of nephrogenesis within the metanephros starts in the subcapsular nephrogenic zone and maturation progresses along the cortico-medullary axis. Therefore, all stages of nephron development, including the metanephrogenic blastema, renal vesicles, comma- and S-shaped bodies and maturing glomeruli, can be found in a single section. In kidneys of E17.5 embryos, β-galactosidase activity was dependent on doxycycline (), and it was seen within all stages of tubular epithelial development later than the S-shaped body stage and in all developing collecting duct cells (). Metanephrogenic blastema, comma- and S-shaped bodies, and maturing glomeruli did not stain positively for β-galactosidase ().
Time course of transgene induction
We evaluated the kinetics of transgene induction in Pax8-rtTA/LC-1 double-transgenic mice (n = 3) using a tetracycline-responsive luciferase reporter gene and noninvasive bioluminescence imaging.
In the absence of doxycycline, no luciferase activity was found (). However, 2 h after the start of induction, luciferase activity (>3 × 104 relative light units (RLU); sampling time, 180 s) could be detected, and it increased by more than tenfold between 2 and 6 h (). Colored overlay pictures revealed that the kidneys were the major source of the collected bioluminescence signal ().
Figure 4 Time course of transgene induction in Pax8-rtTA/LC-1–transgenic mice upon doxycycline administration. (a) Time course of luciferase induction in three individual double-transgenic mice (exposure time, 180 s). Signal intensities are plotted relative (more ...)
To study reversibility of transgene expression, we assayed bioluminescence in three groups of mice over 4 weeks (n = 4 per group). One group was given doxycycline continuously, the second and third group for periods of 5 d and 2 d, respectively. Again, the luminescent signal rapidly developed and reached maximum levels (>1 × 107 RLU; sampling time, 1 s) within 3 d in each group (). The signal remained high as long as the mice were given doxycycline (group 1, ) but dropped sharply when treatment was stopped (groups 2 and 3, ). Multiple cycles of induction were feasible.
PKD and renal cancer induced by overexpression of c-MYC
Mice overexpressing the proto-oncogene product c-MYC in renal epithelial cells develop morphological features similar to autosomal dominant polycystic kidney disease10
, but with a markedly earlier onset. To generate a model of adult onset of PKD, we crossed Pax8
-rtTA mice with TetO-MYC mice, which express c-MYC under control of the Ptet11
Pax8-rtTA/TetO-MYC double-transgenic mice were healthy, with no detectable phenotype. The same mice treated with doxycycline, however, rapidly developed cysts in all renal tubular compartments (), leading to renal failure within 3–4 months of treatment. We also observed adenomas (), glomerular cysts () and renal cell carcinomas () in this model.
Figure 5 Renal disease models. (a–d) Histological analysis of polycystic kidneys showing different stages of malignant progression induced in Pax8-rtTA/tetO-MYC double-transgenic mice. Doxycycline was continuously administered to 3-month-old Pax8-rtTA/tetO-MYC (more ...)
Renal fibrosis caused by overexpression of Tgf-β1
Tgf-β1 has a key role in the onset of renal fibrosis and progression of chronic renal disease. To generate a mouse model of Tgf-β1 production in renal tubular epithelial cells, we bred Pax8
-rtTA mice with TetO–Tgf-β1 mice12
. Upon induction with doxycycline, double-transgenic mice became severely ill within only a few days. The kidneys were not yet severely affected in these mice, but the toxicity may result from high systemic levels of Tgf-β1, as similar toxicity has been reported with the Tet-off system in the liver13
. To avoid lethality, we applied a protocol of discontinuous treatment. Mice were given doxycycline for only 2 d and then pure water for 5 d a week before they were induced again. This kind of treatment leads to maximum serum levels of 504 ng ml−1
(± 68 ng ml−1
) doxycycline and complete clearance (<5 ng ml−1
) within 24 h of doxycycline deprivation (assayed in nontransgenic C57BL/6 mice; data not shown). We carried out ELISAs to determine Tgf-β1 abundance. After 2 d of treatment with doxycycline, we found high amounts of Tgf-β1 in Pax8
-rtTA/TetO–Tgf-β1 double-transgenicmice (plasma: 325.2 ng ml−1
(± 55.1 ng ml−1
= 3; kidney: 4.5 ng per mg protein (± 0.4 ng per mg protein), n
= 3), whereas Tgf-β1 was undetectable (<100 pg ml−1
) in single-transgenic control mice or in double-transgenic mice before induction. Pax8
-rtTA/TetO–Tgf-β1 mice treated for six cycles with doxycycline showed a distinct renal fibrosis phenotype, as we could show by collagen I immunohistochemistry (). Doxycycline alone did not have any adverse effect on renal histomorphology or function even after ten cycles of treatment in control mice (data not shown).
Severe PKD in conditional knockout mice of Tsc1
We tested the ability of Pax8
-rtTA mice to drive the expression of Cre recombinase in a conditional knockout model of Tsc1
, one of the many genes that are embryonically lethal when constitutively inactivated in conventional knockout mice14,15
-rtTA mice were interbred with LC-1 (ref. 8
) and Tsc1flox/flox
) mice to generate Pax8
triple-transgenic mice. Treatment with doxycycline in these experiments was confined to pregnancy and was stopped upon delivery.
In the absence of doxycycline, Pax8-rtTA/LC-1/Tsc1flox/flox mice were healthy, fertile and showed no obvious phenotype. When, however, Pax8-rtTA/LC-1/Tsc1flox/flox mice were exposed to doxycycline in utero, newborn mice showed massive hyperproliferation of proximal and distal tubules and collecting duct epithelial cells, which resulted in fulminant cyst formation. These mice died 3–4 weeks after birth with giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease ().