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Int J Biol Sci 2010; 6(1):68-79. doi:10.7150/ijbs.6.68 This issue Cite

Research Paper

Keratin promoter based gene manipulation in the murine conducting airway

Stephen P. Malkoski^{1,3 ✉}, Timothy G. Cleaver¹, Shi-Long Lu², Jessyka G. Lighthall^*, Xiao-Jing Wang^2,3✉

1. Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver Health Sciences Center, Aurora CO, USA
2. Department of Otolaryngology, University of Colorado Denver Health Sciences Center, Aurora CO, USA
3. Department of Pathology, University of Colorado Denver Health Sciences Center, Aurora CO, USA
* Current Address: Department of Otolaryngology, Oregon Health & Sciences University, Portland, OR, USA

✉ Correspondence to: Xiao-Jing Wang, MD, PhD, Departments of Pathology, Dermatology and Otolaryngology, 12800 E. 19th Avenue, Research 1 North, Room 5128, Box 8104 Aurora, CO 80045. Phone: 303-724-3001 Fax: 303-724-4730 Email: XJ.Wangedu. Or Stephen Malkoski, MD, PhD, Division of Pulmonary Sciences and Critical Care Medicine and Pathology,12800 E. 19th Avenue, Research 2, C272, Aurora, CO 80045. Phone: 303-724-6058 Fax: 303-724-4730 Email: Stephen.Malkoskiedu More

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: Keratins, Cre recombinase, basal cells

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