Figure 4. Prenatal and postnatal RB ablation occurs throughout the lung epithelium and is present in both Clara and ciliated cells.

PCR analysis on lung DNA from postnatal (PN) day 1 or adult double transgenic mice treated with doxycycline (Dox) during gestation (Prenatal Dox), for 3 weeks at 2–3 months of age (Postnatal Dox) or not treated with doxycycline (No Dox) (A). Control lanes show migration of the recombined (RB^Rec), wild type (RB^WT), and floxed (RB^LoxP) RB alleles. Adult mice were homozygous for RB^LoxP, and day 1 pups were homozygous or heterozygous for RB^LoxP. RB^Rec was detected in the PN day 1 lungs only after doxycycline treatment. In contrast, RB^Rec was detected in adult lungs in the absence and presence of doxycycline treatment. Thyroid stimulating hormone beta subunit (TSHbeta) was amplified in each sample as an endogenous internal control to verify equivalent template DNA quality and quantity. Data is representative of 5 No Dox and 36 Prenatal Dox (15 homozygous and 21 heterozygous for RB^Lox) PN day 1 lungs, and ≥ 3 adult lungs for each treatment group. Varied levels of recombination among samples is due, at least in part, to differences in the relative proportion of conducting airway epithelium represented in the lung tissue used to isolate DNA. Enzymatic staining for β-galactosidase performed on lung sections from adult double transgenic mice haRBoring the ROSA26 reporter locus after prenatal doxycycline treatment (B), postnatal doxycycline treatment (C) or no doxycycline treatment (D). Representative low (top panels) and high (bottom panels) power images are shown. β-galactosidase staining (blue) was epithelial specific and present throughout the conducting airways in all groups indicating active Cre recombinase and thus RB ablation. Epithelial staining in the conducting airways was less uniform after postnatal treatment and no treatment as compared to prenatal treatment (compare B to C and D). Ciliated (arrows) and Clara cells (arrowheads) were stained in all three treatment groups. Data is representative of at least 3 animals for each treatment group. br=bronchiole; Original magnification: 200× (top panels) 1000× (bottom panels)