High-Efficiency Type II Cell–Enhanced Green Fluorescent Protein Expression Facilitates Cellular Identification, Tracking, and Isolation

Abstract

We have developed a transgenic mouse expressing enhanced green fluorescent protein (EGFP) in virtually all type II (TII) alveolar epithelial cells. The CBG mouse (SPC-BAC-EGFP) contains a bacterial artificial chromosome modified to express EGFP within the mouse surfactant protein (SP)-C gene 3' untranslated region. EGFP mRNA expression is limited to the lung. EGFP fluorescence is both limited to and exhibited by all cells expressing pro-SP-C; fluorescence is uniform throughout all lobes of the lung and does not change as mice age. EGFP(+) cells also express SP-B but do not express podoplanin, a type I (TI) cell marker. CBG mice show no evidence of lung disease with aging. In 3 hours, TII cells can be isolated in >99% purity from CBG mice by FACS; the yield of 3.7 ± 0.6 × 10(6) cells represents approximately 25 to 60% of the TII cells in the lung. By FACS analysis, approximately 0.9% of TII cells are in mitosis in uninjured lungs; after bleomycin injury, 4.1% are in mitosis. Because EGFP fluorescence can be detected for >14 days in culture, at a time that SP-C mRNA expression is essentially nil, this line may be useful for tracking TII cells in culture and in vivo. When CBG mice are crossed to transgenic mice expressing rat podoplanin, TI and TII cells can be easily simultaneously identified and isolated. When bred to other strains of mice, EGFP expression can be used to identify TII cells without the need for immunostaining for SP-C. These mice should be useful in models of mouse pulmonary disease and in studies of TII cell biology, biochemistry, and genetics.

Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice

“…Our current and previous (Vanderbilt et al. ) findings with CBG mice support a role for type II cells in differentiation of type I cells after lung injury. However, there is evidence for other candidate progenitor cells.…”

“…, arrow in panels F and G), whereas other cells were squamous in shape, resembling type I cells (arrowheads) and positive for the type I cell marker OTS‐8 as previously reported (Vanderbilt et al. ). CEACAM6 staining was observed in some cuboidal cells and more extensively in flattened epithelial cells (arrowheads, panels e and g) where co‐localization with OTS‐8 (Fig.…”

“…); staining for both type I and type II cell markers in a single cell, which is indicative of a transitional alveolar cell, is not found in normal infant (unpublished) or adult (Vanderbilt et al. ) lung. Although it is not possible to study the time course of CEACAM6 expression in postmortem specimens of human lung, it is likely that lung disease evokes proliferation of CEACAM6 + cells in injured alveoli with subsequent epithelial repair or continuing cell proliferation in more severe, chronic cases.…”

“…In the CBG mouse ~99% of type II cells express EGFP (Vanderbilt et al. ). In uninjured CEABAC/CBG mice, cells positive for EGFP are all cuboidal and apparent co‐localization of EGFP (green) and CEACAM6 (red) staining was occasionally observed as shown in Figure (arrow in panel C); most EGFP + type II cells were negative for CEACAM6 under the conditions used.…”

“…By fluorescence microscopy, lungs of CBG mice express enhanced green fluorescent protein (EGFP) in virtually all type II cells (Vanderbilt et al. ). The Committee on Animal Research of the University of California, San Francisco, approved all studies, and all procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals.…”