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Am J Physiol Lung Cell Mol Physiol (May 9, 2003). doi:10.1152/ajplung.00034.2003
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Submitted on February 4, 2003
Accepted on May 7, 2003

IDENTIFICATION AND ISOLATION OF MOUSE TYPE II CELLS BASED UPON INTRINSIC EXPRESSION OF ENHANCED GREEN FLUORESCENT PROTEIN

Jason M. Roper1, Rhonda J. Staversky2, Jacob N. Finkelstein2, Peter C. Keng3, and Michael A. O'Reilly2*

1 Department of Environmental Medicine, The University of Rochester, Rochester, NY, USA
2 Department of Pediatrics, The University of Rochester, Rochester, NY, USA
3 Department of Radiation Oncology, The University of Rochester, Rochester, NY, USA

* To whom correspondence should be addressed. E-mail: michael_oreilly{at}urmc.rochester.edu.

The unique morphology and cell specific expression of surfactant genes has been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells based upon transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that co-localized by immunostaining with endogenous proSP-C were observed scattered throughout the parenchyma. EGFP was not detected in CCSP-expressing airway epithelial cells or other non-lung tissues. ProSP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence because EGFP is not secreted. Type II cells could also be purified from single cell suspensions of lung homogenates using fluorescence activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared to >95% in the sorted population. As expected for type II cells, ultrastructural analysis revealed the sorted cells contained numerous lamellar bodies. SP-A, SP-B and SP-C mRNAs were detected in the sorted population but not T1{alpha} or CD31 (PECAM), indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.




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