Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein.
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 T1alpha 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.
Dept. of Pediatrics, Box 850, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642, USA.
Jason M
JM
Roper
Rhonda J
RJ
Staversky
Jacob N
JN
Finkelstein
Peter C
PC
Keng
Michael A
MA
O'Reilly
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D018496:Hyperoxia / Q000473:pathology; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D008171:Lung Diseases / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020545:Respiratory Mucosa / Q000166:cytology*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins