Keratin promoter based gene manipulation in the murine conducting airway

Abstract

Systems capable of targeting genetic manipulations to keratin-positive airway basal cells are more poorly developed than systems targeting other airway epithelial cell populations and this has likely hindered development of animal models of diseases such as lung squamous cell carcinoma. Although keratin promoter driven-Cre recombinase constructs are potentially useful for targeting these cells, these constructs have substantially higher activity in the skin and oral epithelium than in the airways. We developed a method for delivering RU486, the conditional activator of Cre recombinase progesterone receptor (CrePR) fusion proteins to the lung and then examined the activity of three keratin-driven CrePR constructs in the conducting airways. We also developed a technique for survival bronchioalveolar lavage on non-ventilated animals to examine the effects of the acetone/oil vehicle required to deliver RU486 to the lung. K5CrePR1 and K14CrePR1 constructs differ only in the keratin promoter used to target CrePR1 expression while K5Cre*PR contains a truncated progesterone receptor designed to reduce RU486-independent Cre activity. While all three constructs demonstrate RU486-inducible Cre activity in the conducting airways, both construct activity and tightness of regulation vary considerably. K5Cre*PR is the most tightly regulated Cre driver making it ideal for targeting somatic mutations to the airway epithelia while K5CrePR1 and K14CrePR1 may be better suited to studying diseases of the conducting airways where gene targeting of keratin expressing cells and their derivatives is desired.

Keywords: Cre recombinase; Keratins; basal cells.

Figure 1

(A) Keratin CrePR mice express a Cre recombinaseprogesterone receptor (PR) fusion protein whose expression is restricted by a K5 or K14 promoter. CrePR is inducible by the progesterone antagonist RU486 but not by endogenous progesterone. Upon induction with RU486, CrePR translocates into the nucleus and excises DNA sequences flanked by Lox P sites. Rosa26 reporter mice contain a lox P-neomycin-lox P sequence between a constitutive promoter and a β-gal reporter hence after Cre recombinase-mediated excision of the neomycin cassette β-gal is constitutively expressed and can be used to monitor Cre recombinase activity. (B) Immunofluoresence for K5/K14 positive basal cells in the human and mouse trachea demonstrates the simpler conducting airway epithelium in the mouse. Minimal background immunostaining was observed when sections were incubated without primary antibody (not shown). The immunostaining pattern is consistent with marking K5/K14 positive basal cells. In the human trachea there are 23.4 ± 1.4 (mean ± SEM) K5 positive cells/100 μm epithelium and 5.6 ± 0.9 K14 positive cells/100 μm epithelium (p<0.05 for K5 vs. K14 positive). In the mouse, there are 13.6 ± 2.6 K5 positive cells/100 μm epithelium and 1.9 ± 0.9 K14 positive cells/100 μm epithelium; in the axial bronchus there are 7.5 ± 2.1 K5 positive cells/100 μm epithelium and 0.25 ± 0.2 K14 positive cells/100 μm epithelium (p<0.05 for K5 positive vs. K14 positive in both murine trachea and axial bronchus).

Figure 2

(A) Excised heartlung block after tracheal instillation of 10μl 0.01% Evans blue dye in 10% acetone/90% sesame oil showing staining in the trachea and lung but not in the esophagus. (B) Evans blue dye instillation to the right lung was performed as described in the methods. (C) Recovery of LLM by survival BAL after tracheal instillation of acetone/sesame oil. Untreated 8-12 week old animals (n=12) underwent BAL with 200μl normal saline as described in Methods. Approximately 95% of recovered cells were macrophages. A separate group of mice (n=15) was treated with 20μl of 20% acetone/80% sesame oil on day 0 then underwent survival BAL on either day 3, 21, or 45 (n=5 for each time point). Three days after treatment there was marked increase in both LLM and neutrophils (bottom left), however, these changes largely resolved by day 45. * p<0.05 vs. untreated control for all cell types; **p<0.05 vs. days 3 and 21 for all cell types.

Figure 3

Cre activity after tracheal RU486 in K5CrePR1Rosa26 mice. (A) K5CrePR1.Rosa26 mice were treated with 100μg tracheal RU486 and β-gal activity assessed 7d later. No β-gal staining was observed in Cre negative monogenic littermates while RU486-inducible Cre activity was seen in K5CrePR1.Rosa26 animals. Representative images are shown; 3-4 mice/group were analyzed for β-gal expression. The dashed line delineates the epithelial basement membrane; the scale bar represents 100μm. Untreated K5CrePR1.Rosa26 mice had 5.3 ± 1.0 (mean ± SEM) recombinant cells/100μm tracheal epithelium while RU486-treated mice had 14.5 ± 4.2 recombinant cells/100μm tracheal epithelium (p=0.05). (B) Co-localization of β-gal staining with K5, p63, and CCSP expression in K5CrePR1.Rosa26 mice treated with tracheal RU486. Arrows indicate co-localization of β-gal and immunohistochemical staining in basal cells (K5+ and p63+) and Clara cells (CCSP+).

Figure 4

Cre recombinase activity in K14CrePR1Rosa26 mice. Mice were treated with 500μg of tracheal RU486 on days 1 and 5 and then assessed for β-gal activity 7d after the last RU486 dose. (A) Cre activity was induced in the trachea and was localized to discrete areas in the tracheal epithelium, however, β-gal staining was also occasionally observed in the lung parenchyma, consistent with rare basal cells in more distal airways. Representative images are shown; 3-8 mice per group were analyzed for β-gal expression. (B) Immunohistochemistry for K5, p63, and CCSP colocalizes with β-gal staining in K14CrePR1.Rosa26 mice treated with tracheal RU486. The scale bar represents 50μm; arrows indicate co-localization of β-gal with K5, p63, and CCSP immunohistochemical staining; 1.3 ± 0.6 recombinant cells/100μm tracheal epithelium were observed in RU486 treated animals compared to 0.1 ± 0.07 recombinant cells/100μm tracheal epithelium in vehicle treated animals (p<0.05).

Figure 5

Inducible and persistent Cre recombinase activity in K5Cre*PRRosa26 mice after tracheal RU486. (A) In mice treated with 500μg of tracheal RU486 on days 1 and 5 and assessed for β-gal activity 7d later, discrete areas of β-gal activity were seen in the trachea. β-gal staining was not observed in the tongue or esophagus of RU486-treated animals (not shown) or in vehicle treated animals. Representative images are shown; 5-10 mice per group were analyzed. (B) β-gal activity colocalized with K5, p63, and CCSP immunostaining in the trachea of RU486-treated K5Cre*PR.Rosa26 mice. The scale bar represents 50μm; arrows indicate co-localization of β-gal and immunohistochemical staining; 0.3 ± 0.16 recombinant cells/100μm tracheal epithelium were observed in RU486 treated mice; recombinant cells could not be found in the tracheal epithelium of vehicle treated animals. (C) Persistent tracheal Cre recombinase activity in K5Cre*PR.Rosa26 mice 24 weeks after tracheal RU486. Although β-gal staining was weaker compared to earlier time points, it still colocalized with K5, p63, and CCSP staining and was not detected in vehicle-treated tissues (top panel). The scale bar represents 50μm; 17.5 ± 0.8 recombinant cells/100μm tracheal epithelium were observed (p<0.05 vs. 7d after RU486).