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Departments of 1Environmental Medicine, 2Pediatrics, and 3Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642
Submitted 4 February 2003 ; accepted in final form 7 May 2003
The unique morphology and cell-specific expression of surfactant genes have
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 on the basis of 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
colocalized by immunostaining with endogenous pro-SP-C were scattered
throughout the parenchyma. EGFP was not detected in Clara cell secretory
protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-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 with >95% of the sorted population. As expected
for type II cells, ultrastructural analysis revealed that the sorted cells
contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected
in the sorted population, but T1
and CD31 (platelet endothelial cell
adhesion molecule) were not, 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.
fluorescence-activated cell sorting; surfactant proteins; transgenic
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