The Nkx3.1CreERT2/+ allele was generated by gene targeting using standard techniques; the Nkx3.1 null mutant mice have been previously described21 (link). R26R-lacZ and Pten conditional mutant mice were obtained from the Jackson Laboratory Induced Mutant Resource; the R26R-YFP mice were provided by Dr. Frank Costantini. All lines were maintained on a hybrid C57BL/6-129/Sv strain background.
Castration of adult male mice was performed using standard techniques. For tamoxifen induction of Cre activity in mice containing Nkx3.1CreERT2/+, mice were administered 9 mg/40 g tamoxifen for 4 consecutive days. For prostate regeneration, physiological levels of testosterone (1.875 µg/hr) were administered for four weeks by subcutaneous implantation of mini-osmotic pumps (Alzet)45 (link). When included, BrdU (100 mg/kg) was administered once daily during the first three days of regeneration. For single-cell transplantation, single YFP+ cells were isolated by mouth-pipetting under epifluorescence illumination from a dissociated prostate cell suspension obtained from castrated and tamoxifen-induced Nkx3.1CreERT2/+; R26R-YFP/+ mice. A single YFP+ cell (or YFP− cell as a control) was recombined with 2.5 × 105 rat urogenital sinus mesenchyme cells in a 10 µl collagen pad, followed by transplantation under the kidney capsule of nude mice and harvesting after 10–12 weeks.
Cryosections were stained with primary antibodies as listed inSupp. Table 5 , and counterstained with TOPRO3 or DAPI (Invitrogen/Molecular Probes). Secondary antibodies were labeled with Alexa Fluor 488 , 555, or 594 (Invitrogen/Molecular Probes). Immunofluorescence staining was imaged using a Leica TCS5 spectral confocal microscope. Cell counting was performed manually using confocal photomicrographs with at least three animals for each experiment or genotype analyzed.
Castration of adult male mice was performed using standard techniques. For tamoxifen induction of Cre activity in mice containing Nkx3.1CreERT2/+, mice were administered 9 mg/40 g tamoxifen for 4 consecutive days. For prostate regeneration, physiological levels of testosterone (1.875 µg/hr) were administered for four weeks by subcutaneous implantation of mini-osmotic pumps (Alzet)45 (link). When included, BrdU (100 mg/kg) was administered once daily during the first three days of regeneration. For single-cell transplantation, single YFP+ cells were isolated by mouth-pipetting under epifluorescence illumination from a dissociated prostate cell suspension obtained from castrated and tamoxifen-induced Nkx3.1CreERT2/+; R26R-YFP/+ mice. A single YFP+ cell (or YFP− cell as a control) was recombined with 2.5 × 105 rat urogenital sinus mesenchyme cells in a 10 µl collagen pad, followed by transplantation under the kidney capsule of nude mice and harvesting after 10–12 weeks.
Cryosections were stained with primary antibodies as listed in
