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.

Keywords: alveolar epithelium; alveolar type II cells; bacterial artificial chromosome; enhanced green fluorescent protein; surfactant protein C.

Figure 1.

(A) Expression of the enhanced green fluorescent protein (EGFP) transgene is limited to alveolar epithelial type II (TII) cells in SPC-BAC-EGFP (CBG) mice. (A, B, and D) EGFP⁺ TII cells (green, arrows) and the apical plasma membranes of alveolar epithelial type I cells (red) labeled with anti-mouse podoplanin (OTS8) (34)/anti-hamster IgG-Alexa 594. (A) Combined red and green channels showing that the entire epithelium can be visualized; there are no discontinuities in the fluorescence. (B) Red channel shows that TII cells are not decorated with mouse podoplanin (arrows). (C) Matching phase contrast image showing lucent lamellar bodies (arrows) in TII cells. (D and E) Lower-magnification paired immunofluorescence and phase contrast images showing lack of either EGFP expression or podoplanin staining in airways and blood vessels (BV). The alveolar epithelial surface expresses either EGFP or podoplanin. (F–N) Colocalization of EGFP with surfactant protein (SP)-B and SP-C. (F–H) The distribution of pro–SP-C is similar in wild-type (F) and CBG mice (G and H). (I–K) Phase contrast (I) and fluorescence images showing colocalization of SP-C (J and K) with EGFP (K). (L–N) TII cells can be clearly identified at higher magnification by the presence of phase-lucent lamellar bodies (LB) (arrows) and colocalization with SP-B (M and N) and EGFP (N) in TII cells. AB, antibody; WT, wild type.

Figure 2.

FACS isolation of EGFP⁺ cells from CBG lungs and persistence of EGFP expression in culture. (A) Scattergram of CBG mouse lung cells sorted for EGFP fluorescence, either without propidium iodide (PI) or with PI. Fraction 1 contained cells with very low levels of fluorescence; fraction 2 cells exhibited low fluorescence; cells in fraction 3 exhibited fluorescence approximately 100-fold higher than fraction 2. Scattergram of FACS sorted cells after the addition of PI, which stains dead/damaged cells, demonstrating that fraction 2 consists of dead/damaged cells. (B–G) Cytospins of cells from each of the three fractions showing paired phase contrast and fluorescence images. Fraction 1 (B, E) contained morphologically heterogeneous cells that were mostly not fluorescent. Fraction 2 (C, F) contained cell ghosts of very low fluorescence. Fraction 3 (D, G) contained uniform round cells of high fluorescence. (H–J) Higher-magnification views of cells in fraction 3 demonstrating that these cells exhibit EGFP fluorescence (H) and contain immunoreactive SP-B (I). (J) A merged SP-B/EGFP image is shown. EGFP⁺ cells all express SP-B. Cells from fraction 3 were placed in tissue culture and cultured on glass cover slips coated with fibronectin. (K–P) Paired phase contrast and fluorescence images of TII cells showing that, although cells flatten, attenuate, and spread with time in culture, EGFP fluorescence persists. Exposures were taken with constant exposure time, showing that fluorescence is weaker in culture as cells flatten and thin. (Q–S) Cells maintained for up to 17 days still have detectable fluorescence. In these images, camera exposure time was set on “automatic”; although fluorescence intensity decreased with time in culture, it can easily be detected. (T) Time course of mRNA expression in culture showing that over a 7-day period, mRNA levels for both SP-C and EGFP fall by approximately 104-fold. (U) Time course of protein expression showing that SP-C and EGFP protein levels fall by approximately 5-fold over a week.

Figure 3.

TII cell shape changes in areas of lung injury after treatment with bleomycin, and cells colocalize with podoplanin. Paired immunofluorescence and phase contrast images showing immunostaining for OTS8 (red) and EGFP fluorescence (green). EGFP expression can be used to track changes in cell shape in injured lung after treatment with bleomycin. Four days after bleomycin instillation, TII cells appear to flatten (A–C), some more than others. (D–F) In some areas, one can detect colocalization of EGFP and OTS8 (arrows), which is not seen in uninjured mice.