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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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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
Although mice are advantageous for manipulating genes, they have not been useful for isolating type II cells for ex vivo study. In contrast, rat and rabbit type II cells have successfully been isolated using velocity centrifugation through a gradient of albumin (8, 10). Isolation of mouse type II cells by this method has been less successful, because airway Clara cells, which are extremely abundant in mice, frequently contaminate the preparations (7, 18). Relatively pure populations of mouse type II cells have been obtained by dispase with agarose instillation and laser-flow cytometry, but it was necessary to label cells with lipid-soluble phosphine fluorescent dye (14). High yields have also been obtained when flow sorting was replaced with panning on plates containing anti-CD32 and anti-CD45 (5, 29). Although epithelial cells are readily obtained with this method, we found that type II cell purity varied between preparations (unpublished observations). Given that the strength of the mouse model is its ability to be genetically manipulated, it would be advantageous to develop new methods by which type II cells could be routinely identified and isolated from genetically defined strains under different experimental conditions.
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria fluoresces brightly when exposed to ultraviolet or blue light (34). The active chromophore is encoded within the primary translation product and does not require enzymatic digestion for activity. A mutant form of GFP has been generated with a red-shifted peak that fluoresces 35 times more intensely than wild-type GFP. This enhanced GFP (EGFP) has been introduced into mammalian cells in vitro and in vivo (9, 13, 24, 32, 37). Fluorescence microscopy and fluorescence-activated cell sorting (FACS) have been used to visualize and purify EGFP-expressing cells from transgenic mouse thymus, bone marrow, and cardiac myocytes (9, 24, 37). On the basis of these studies, we generated a line of mice in which EGFP was expressed in type II epithelial cells under control of the human surfactant protein (SP)-C promoter. This promoter targets genes specifically to the respiratory epithelium (11, 12). Using these mice, we demonstrate that type II cells may be visualized in real time and isolated on the basis of their endogenous green fluorescence.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Tail snips were obtained from potential founders and their progeny at weaning and used to isolate genomic DNA (19). DNA (10 µg) was digested with EcoRI and separated on 0.8% agarose gels. DNA was transferred to Nytran and hybridized with the EGFP cDNA radiolabeled with 32P (3,000 Ci/mmol; New England Nuclear, Boston, MA). Hybridized blots were washed in 1% bovine serum albumin, 40 mM sodium phosphate, and 2 mM EDTA before visualization on a PhosphorImage screen (Molecular Dynamics). Copy number was determined by comparing transgene DNA band intensity with genomic equivalents of pEGFP plasmid.
Analysis of transgene expression in whole lungs. Adult (8-12 wk old) mice were exposed to room air or >95% oxygen (hyperoxia) for 72 h (25). Animals were euthanized by injection with pentobarbital sodium (100 mg/kg ip). Lungs were inflation fixed through the trachea for 15 min with 10% neutral buffered formalin at 10 cm of pressure. Lungs were removed and further fixed for 12 h in the same fixative. Individual lobes were removed and embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA) before they were frozen in liquid nitrogen. Frozen sections (6 µm) were prepared from these lobes using a cryostat and stored at -80°C. Slides were equilibrated to room temperature and washed free of optimum cutting temperature compound using Tris-buffered saline before coverslips were applied, and the slides were visualized. Additional lobes were dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin. Paraffin sections (4-µm) were immunostained with rabbit anti-Clara cell secretory protein (CCSP) serum (28) or rabbit anti-pro-SP-C serum (Chemicon International, Temecula, CA). Paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through graded ethanol and water. Sections for CCSP immunostaining were incubated overnight in primary antibody and detected with Texas red dye-conjugated donkey anti-rabbit IgG (Jackson Labs, West Grove, PA). Sections for pro-SP-C immunostaining were subjected to antigen retrieval (AR) by boiling in 50 mM Tris, pH 9.5, incubated overnight in primary antibody, subjected to tyramide signal amplification (TSA) using a TSA-biotin system, and detected with streptavidin-Texas red (New England Nuclear). EGFP was detected with anti-EGFP serum (Clontech) and 3,3'-diaminobenzidine precipitation using previously described methods (25) or in sections subjected to AR with an FITC-conjugated goat anti-EGFP antibody (Novus Biologicals, Littleton, CO). Sections were immersed in 4',6-diamidino-2-phenylindole, and fluorescence was visualized with a fluorescence microscope (model E800, Nikon, Melville, NY). Images were captured with a digital camera (SPOT-RT, Diagnostic Instruments, Sterling Heights, MI).
Protein expression was also determined by Western blot analysis. Tissues
were homogenized in lysis buffer containing protease inhibitors
(25). Proteins were separated
on 12% polyacrylamide gels and transferred to polyvinylpyrolidine membrane.
Membranes were blocked overnight at 4°C in 5% nonfat dry milk before
incubation in anti-EGFP serum (Clontech) or 
-actin serum (Sigma
Chemical, St. Louis, MO). Immune complexes were detected by chemiluminescence
(Amersham, Arlington Heights, IL) and visualized by exposure to Kodak Bio-Max
film.
Isolation of type II cells by FACS. Mice were euthanized by injection with pentobarbital sodium (100 mg/kg ip). The renal vessels of the left kidney were isolated and lacerated to reduce blood volume. The pulmonary capillaries were perfused with 10 ml of sterile saline to remove erythrocytes. The lungs were dissected free of the thoracic cavity and rinsed in sterile saline. Two different methods were used to dissociate epithelial cells from the lung. One method involved dissociating the lungs with surgical scissors and chopping them into a fine paste using an automatic tissue chopper. Minced tissues were incubated in 10 ml of protease cocktail containing 0.5 mg/ml collagenase, 0.5 mg/ml pronase, 0.4 mg/ml DNase, and 0.006% sodium bicarbonate in Hanks' balanced salt solution for 1 h at 37°C. Dissociated tissue was then filtered through a sterile 100-µm nylon cell strainer, and the single-cell suspension was pelleted at 300 g and 4°C. Pellets were resuspended in a suitable volume of PBS, and cell counts were obtained using a Coulter counter. A second method involved instillation of perfused lungs with dispase and then with low-melt agarose before preparation of single-cell suspensions (5). Cell suspensions were successively filtered through 100- and 40-µm cell strainers and finally through 25-µm nylon gauze. Single-cell suspensions were then pelleted by centrifugation at 300 g for 5 min at 4°C. Cells obtained from transgenic mice (10 per experiment) were pooled and resuspended in 10 ml of DMEM with 0.5% FBS and 25 mM HEPES. Cells obtained from nontransgenic mice (1 per experiment) were resuspended in 2 ml of medium. Presort cell counts were obtained using a Coulter counter.
Green fluorescent (EGFP) type II cells from dissociated lung tissues were
isolated using a cell sorter (B-D FACSVantage SE, Becton Dickinson
Immunocytometer Systems, Palo Alto, CA). A two-step sorting procedure was used
in our study to isolate the small population of EGFP-expressing cells with
high purity and yield. Single-cell suspensions were sorted first with the
"enrich" mode to collect every EGFP-positive cell. This procedure
resulted in enrichment of the EGFP-positive cells from 
1% to 26%. The
enriched cell populations were sorted again with the "normal-R"
mode to achieve final purity of 95%. Cellular EGFP was excited by an
argon ion laser emitted at the wavelength of 488 nm, and the fluorescence was
collected after a 530 ± 30 nm band-pass filter. A two-parameter sorting
window (forward light scattering and EGFP fluorescent intensity) was used to
define the EGFP-positive cell populations. The cells were sorted through a
flow chamber with an 80-µm nozzle tip under 12 psi sheath fluid pressure.
The sorted cells were collected into 15-ml conical tubes filled with sterile
media for morphological and biochemical assays. On average, 2.5 h were
required to prepare a single-cell suspension of 1.5 x 108
cells from 10 mice. The enrich-mode sort was carried out at a rate of 10,000
cells/s, and completion requires 4.5 h. The normal-R mode sort occurred
at a rate of 1,000 cells/s, and completion requires 1 h.
Isolated cells were cytospun onto glass slides, fixed in a 2.5% phosphate-buffered glutaraldehyde, pH 7.4, and post-fixed in a 1.0% phosphate-buffered osmium tetroxide. The slides were passed through a graded series of ethanol to 100% and infiltrated with liquid Spurr epoxy resin and embedded on the glass surface with inverted capsular molds containing fresh resin. After polymerization at 70°C, the hardened capsules were then popped off the glass slide by dipping into liquid nitrogen. The "popped-off" capsules were examined with a light microscope to determine the area to be trimmed and thin sectioned with a diamond knife onto 200-mesh copper grids. The grids were contrasted with aqueous uranyl acetate for 10 min and lead citrate for 15 min, examined, and photographed with a transmission electron microscope (model 7100, Hitachi).
Genotypic analysis by RT-PCR. Gene expression was determined by
RT-PCR using total RNA isolated by phase-lock gel microcentrifuge tubes (5
Prime-3 Prime, Boulder, CO)
(25). cDNA templates were
synthesized at 42°C for 30 min using RT and oligo(dT) primers
(Perkin-Elmer, Foster City, CA). All products were amplified with 15 µM
primers and cycling 35 times at 94°C for 1 min, 51°C (SP-C), 57°C
(SP-A and T1-) or 60°C (EGFP, SP-B, and aquaporin-5) for 1 min, and
72°C for 1 min. Primers for EGFP were
5'-ATGGTGAGCAAGGGCGAGGAGCTG-3' and
5'-CTTGTACAGCTCGTCCATGCCGAG-3', which amplified a 716-bp product.
Primers for SP-A were 5'-AAAGGGGGCTTCCAGGGTTTCCAGC-3' and
5'-ATTCCTCGGGGCAGCAATGTGG-3', which amplified a 224-bp product.
Primers for SP-B were 5'-TGTCTACCTGCCCCTGGTTATTG-3' and
5'-GCATCCTCAGTGGAACATCGG-3', which amplified a 471-bp product.
Primers for SP-C were 5'-CATGAAGATGGCTCCAGA-3' and
5'-TTTGTGATAGGATCCCCC-3', which amplified a 400-bp product.
Primers for aquaporin-5 were 5'-GGAGGTGTGTTCAGTTGCCTTC-3' and
5'-CTCAGCGAGGAGGGGAAAAG-3', which amplified a 684-bp product.
Primers for T1 were 5'-TACTGGCAAGGCACCTCTGG-3' and
5'-TCTGCGTTTCATCCCCTGC-3', which amplified a 200-bp product.
Primers for CD31 (platelet endothelial cell adhesion molecule) were
5'-AAGCGAAGGATAGATAAGACCTC-3' and
5'-CAGGATGGAAATCACAACTTCA-3', which amplified a 995-bp
product.
Statistical analysis. Where appropriate, values are means ± SD. Group means were compared by ANOVA using Fisher's procedure post hoc analysis, and P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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20% of progeny from
founder 28a, indicating that her germ cells were mosaic for the
transgene. Her F1 progeny transmitted the transgene in typical Mendelian
fashion. Unfortunately, EGFP mRNA and protein were not detected, so this line
was not examined further. Founder 67 transmitted the transgene in
50% of his progeny, with typical Mendelian inheritance by the second
generation. Recently, homozygous (transgenic on both chromosomes) mice have
been obtained. Transgenic haplozygous (transgenic on one chromosome with no
complementary allele) progeny contained approximately three to five copies of
the transgene integrated as head-tail concatamers
(Fig. 1B). Because
EGFP mRNA was detected in their lungs (data not shown), these haplozygous mice
were studied further.
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EGFP expression in transgenic mice did not affect fecundity, Mendelian
inheritance, or health of the mice. Nor have signs of respiratory distress or
abnormal histopathology been observed in older mice. EGFP expression was
assessed in a number of tissues by Western analysis. EGFP was readily detected
in lungs of transgenic, but not nontransgenic, littermates
(Fig. 2). As predicted for the
lung-specific expression of the SP-C promoter, EGFP was also not detected in
heart, intestine, kidney, liver, or brain of transgenic mice. The expression
of -actin confirmed that all lanes contained protein.
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Several methods were used to detect cells expressing EGFP. Immunohistochemistry was initially used to localize EGFP expression in transgenic and nontransgenic lungs (Fig. 3, A and B). EGFP-positive cells were restricted to cells in corners of alveoli, rather than along alveolar walls. Under higher-power resolution, they exhibited cuboidal and granular morphology, consistent with the type II cell phenotype (inset, Fig. 3A). Staining was not detected in airway epithelium, fibroblasts underlying airway, endothelial cells surrounding blood vessels, or any cells of the nontransgenic lung. This pattern of expression was confirmed by visualization of intrinsic green fluorescence in frozen sections prepared from lungs of trangenic and nontransgenic mice (Fig. 3, C and D). To confirm EGFP expression in type II cells, tissue sections were immunostained with anti-pro-SP-C and visualized using TSA and streptavidin-Texas red conjugate. Sections were then stained with FITC-conjugated anti-EGFP serum, because endogenous EGFP fluorescence is extinguished by the AR method required for pro-SP-C staining (Fig. 3, E and F). Cells with green and red fluorescence were readily detected, indicating that EGFP-expressing cells also expressed pro-SP-C. Although all EGFP-positive cells expressed pro-SP-C, all type II cells did not express detectable levels of EGFP. EGFP cells also colocalized with pro-SP-B-expressing cells (data not shown). Because the human SP-C promoter can target transgenes to airway Clara cells, EGFP expression was also compared with CCSP expression. Intrinsic green fluorescence was used instead of EGFP immunostaining, because, unlike pro-SP-C, CCSP staining does not require AR. Red CCSP fluorescence was observed in terminal airway epithelial cells that did not overlap with intrinsic green fluorescence (Fig. 3, G and H). These findings indicate that, in this particular line of mice, EGFP expression is restricted to type II epithelial cells with little to no expression in bronchiolar epithelium.
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EGFP fluorescence is retained during lung injury. SP expression is markedly altered when lungs are injured. For example, hyperoxia (>95% oxygen) stimulates SP-B mRNA and protein expression in bronchiolar epithelium while diminishing expression in alveolar type II cells (16, 36). Although the mechanism is unknown, decreased protein expression in type II cells is associated with mRNA loss and, presumably, failure to maintain intracellular levels of precursor protein that is secreted along with surfactant lipids. Because EGFP is not secreted, we hypothesized that it would be retained during hyperoxia, thereby permitting identification of type II cells when SP expression is lost. Transgenic mice were exposed to room air or >95% oxygen for 72 h, and pro-SP-C and EGFP immunostaining were compared (Fig. 4). Green EGFP-positive type II cells were detected in lungs exposed to room air as a yellow-green color because of their colocalization with red pro-SP-C immunostaining. Although EGFP-positive cells were readily detected in hyperoxic lungs, red pro-SP-C immunostaining was markedly diminished throughout the parenchyma. Compared with room air-exposed tissues, the EGFP-positive cells exhibited a bright green fluorescence due to the lack of red pro-SP-C staining. Thus EGFP protein expression is retained during oxygen-induced lung injury and can be used to identify type II cells that have diminished SP expression.
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Isolation of type II epithelial cells on the basis of green fluorescence. Previous studies have shown that intact cells expressing EGFP can be isolated by FACS when excited under ultraviolet light (9, 24, 37). Two different protease cocktails were used to prepare single-cell suspensions that were visualized by fluorescence microscopy. A rare fluorescent cell was detected when lungs were dissociated with pronase and collagenase (data not shown). This suggested that these proteases did not effectively remove type II cells from their matrix. In contrast, brightly fluorescent cells were frequently seen when dispase and agarose were instilled. This confirmed previous studies showing that this method efficiently dissociates epithelial cells from lung tissue (5, 14). Because all cells have low intrinsic fluorescence, especially conducting airway epithelial and red blood cells, the overall fluorescence was initially determined by FACS using dissociated cells from a nontransgenic lung (Fig. 5). After we set an upper-limit gate that considered 0.053 ± 0.075% (M2 region in Fig. 5) of the cells highly fluorescent, cells from a transgenic lung were sorted using the normal-R mode and a 1.5-drop sort envelope. We discovered that a long sorting time was required and very few EGFP-positive cells were collected. Furthermore, overall purity varied between experiments because of contaminating red blood cells. To improve the purity and recovery of sorted cells, a two-step sorting procedure was established. Cells were first sorted using the enrich mode to ensure that every EGFP-positive cell was collected. Assessment of the fluorescence intensity showed that the first-step sorting increased the percentage of EGFP-positive cells from 1.12 ± 0.32% (M2 region in Fig. 5B) to 26.55 ± 1.07% (M2 region in Fig. 5C). The smaller fluorescent peaks in Fig. 5C comprised mostly red blood cells, which were discarded. Enriched EGFP-positive cells were resorted using the normal-R mode and a three-drop sort envelope under more stringent conditions (M3 = 20.79 ± 1.83%). As expected for the heterocellular nature of lung tissue, forward light scatter revealed that the presorted population exhibited a wide variation in cell size (Fig. 6). Although multiple cell sizes were present in the discarded EGFP-negative population, the EGFP-positive population exhibited a uniform cell size consistent with enrichment of a single cell type.
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Pre- and postsorted cells were visualized by fluorescence microscopy. An
occasional brightly fluorescent green cell was observed in the presorted
population (Fig. 7). Dispase
and agarose were used to obtain 2,000,000 cells per lung, of which 2.5
± 0.71% (n = 3) were highly fluorescent. The higher percentage
of fluorescent cells counted by eye than gated by FACS (1%, region M2 in
Fig. 5B) represents
the more stringent criteria used by the laser in the cell sorter. In contrast,
94 ± 6.8% (n = 3) of the postsorted cells were highly
fluorescent as assessed by fluorescence microscopy. Approximately 50-100,000
fluorescent cells were obtained per lung, which could vary depending on the
rate at which cells passed through the FACS. Ultrastructural analysis of
sorted cells revealed that they contained lamellar bodies, characteristic of
type II epithelial cells (Fig. 7,
C and D).
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RT-PCR was used to confirm enrichment of type II epithelial cells in the
sorted population. With the exception of EGFP, primers were designed to bridge
introns so that amplification of genomic DNA could be distinguished from RNA.
Amplification of RNA-derived products was also confirmed by failure to amplify
EGFP when RT was omitted from the reaction
(Fig. 8). SP-A, SP-B, SP-C,
aquaporin-5, T1, CD31, and CCSP were detected in RNA isolated from
whole lung. As expected, SP-A, SP-B, and SP-C were detected in the sorted
cells, consistent with their expression by type II cells. The type I
epithelial cell gene T1 and the endothelial cell gene CD31 (platelet
endothelial cell adhesion molecule) were not detected in the sorted cells.
Interestingly, aquaporin-5 was detected in some experiments, but only when
most of the RT-PCR product was loaded onto the gel (not shown). Because
aquaporin-5 was not always detected, it is likely to be due to the presence of
a contaminating cell type or occasionally expressed by type II cells. Clara
cells are most likely the predominant contaminant, because CCSP was faintly
detected in the sorted population, even though EGFP did not colocalize with
cells expressing CCSP.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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2,000,000 cells from
which 50-100,000 fluorescent type II cells were obtained. Although transgene
expression was not ascertained in the discarded cells, nearly all
EGFP-expressing cells are calculated to have been positively selected for.
Higher yields per mouse will require the generation of additional lines with a
greater percentage of type II cells expressing EGFP. Even with these
limitations, this study established that type II cells can be tracked during
lung injury or isolated by FACS simply on the basis of transgenic expression
of EGFP.
The human SP-C/EGFP mice were developed with the intention that the
intrinsic nature of EGFP-mediated fluorescence might allow one to observe and
isolate type II cells in real time. Taking advantage of the targeting
capabilities of the human SP-C promoter and the knowledge that other lines of
mice have been generated that express EGFP, we successfully generated a line
of mice from which fluorescent type II cells can be identified and isolated.
Surprisingly, EGFP expression recapitulated endogenous SP-C expression, in
that it was restricted to the parenchyma. Previous studies revealed that the
human promoter drives ectopic expression of chloramphenicol acetyltransferase
(CAT) in bronchiolar epithelium and in alveolar type II cells
(12). A detailed deletion
analysis of the promoter revealed that ectopic bronchiolar expression was
dependent on sequences between -1910 and -215, with alveolar expression being
dependent on more proximal sequences
(11). Although the proximal
region provided appropriate expression to type II cells, the proportion of
type II cells expressing CAT varied between individual lines of mice. As
pointed out by Glasser et al.
(11), variation in transgene
expression has also been reported for the -globin promoter. On the basis
of studies with the -globin promoter, Glasser et al. speculated that the
chromatin environment around the integration site influences the proportion of
type II cells that express the SP-C/CAT transgene. Such a scenario would be
consistent with the relatively low proportion of type II cells that express
EGFP in our line of mice. In the same manner, the integration site may have
blocked activity of the distal elements responsible for the ectopic expression
typically seen in bronchiolar epithelium. The Southern blot analysis in
Fig. 1 indicates that the
SP-C/EGFP transgene did not undergo a gross deletion or inversion, which might
explain why bronchiolar expression is not detected. In some ways, the lack of
bronchiolar expression in this line of mice is a benefit for those interested
in exclusively tracking type II cells.
Even though high levels of EGFP can be toxic, it is unlikely to explain why
only some type II cells express the protein. First, lung pathology is normal,
and the mice appear to be unaffected by the transgene. Second, propidium
iodide exclusion revealed that viability of the isolated cells was 94.2
± 0.9% (n = 3). Third, transgenic mice have been created in
which EGFP is expressed in the lung and other tissues under control of the
chicken -actin promoter and cytomegalovirus enhancer
(24). Furthermore, EGFP has
recently been expressed throughout the lung or in specific epithelial
populations depending on when Cre-mediated recombination occurred during
embryogenesis (26). Although
it is highly speculative, EGFP may be expressed only in a subpopulation of
type II cells that are distinguished by factors other than expression of
surfactant genes. Evidence that supports this hypothesis is as follows: weak
expression of aquaporin-5 was detected in some experiments, even though
T1 was not. Interestingly, aquaporin-5 was faintly detected in one of
the sorting experiments where 98% of the sorted cells expressed EGFP. The fact
that T1 has never been detected in the sorted population suggests that
type I cells were successfully excluded. Normally, alveolar type II cells do
not express aquaporin-5 (22).
However, aquaporin-5 has been observed in hyperplastic type II cells as a
result of conditional expression of fibroblast growth factor-7, indicating
that type II cells have the capacity to express this gene
(33). The finding that
aquaporin-5 is occasionally detected in the sorted cells suggests that some or
all type II cells may express low levels of this gene. Genotypic analysis of
these cells using microarray technology may someday reveal whether
subpopulations of type II cells exist.
A number of different methods have been used previously to isolate type II
cells. One method involved purification by velocity sedimentation
(8,
10,
18). Although type II cell
purity approximated 
80% in rats and rabbits, yields from mice rarely reach
65%. Alternatively, type II cells have been isolated by flow cytometry on the
basis of their size, shape, and lipid content using phosphine fluorescence
(14,
20). Purity was improved by
dissociating lungs with dispase and gating against macrophages labeled with
fluorescent lectins. With these methods, 1,000,000-2,000,000 type II
cells per mouse were obtained at >90% purity. The present study also used
dispase and FACS to purify mouse type II cells. Unlike previous flow studies
that used fluorescent dyes to label lipids, type II cells were selected on the
basis of their expression of EGFP. Even though yields were considerably lower
than those from other published methods, type II cell purity was extremely
high. Indeed, only 1 of 100 cells examined by electron microscopy did not
contain lamellar bodies, a key characteristic of type II cells. Moreover, RNA
was purified and used successfully to genotype enrichment of genes expressed
by type II cells. Expression of the CCSP message in the sorted population
indicates that Clara cells, which did not express EGFP, may contaminate the
sorted population. Increasing the sorting stringency will reduce this
population, albeit at the expense of reducing the yield. To our knowledge,
this is the first time that type II cells were not isolated on the basis of
predetermined characteristics of cell shape, size, or lipid content. As such,
a uniform population of type II cells was purified solely on the basis of
their ability to express a single gene.
In addition to isolating cells, this line of mice can also be used to follow type II cells in real time. As shown in Fig. 4, EGFP may be used to identify type II cells during hyperoxia when SP expression diminishes. We have recently been able to isolate fluorescent type II cells from oxygen-exposed lungs for studies on molecular oxidative damage. Thus these isolation methods will be an invaluable new tool for distinguishing and isolating type II cells from animals exposed to a number of different inhaled pollutants. EGFP expression may also be followed in real time as cultured type II cells differentiate into type I cells (3, 4). This process is thought to mimic the normal differentiation events that occur during embryogenesis or remodeling of the injured lung (1, 2). It will be interesting to determine whether EGFP fluorescence can be maintained or restored by altering culturing conditions that allow differentiation of type I-like cells to a type II phenotype (6, 30, 31). Branching morphogenesis has been studied in explant cultures. This process is thought to involve inductive interactions between mesenchyme and epithelium (15). A previous study followed ureteric bud development in three dimensions using EGFP-mediated fluorescence and confocal microscopy (32). Similar studies may now be applied to lung branching morphogenesis. EGFP fluorescence may also be used to follow SP-C transcriptional activity under various experimental conditions, such as during organogenesis or type II cell injury (21, 23). By first backcrossing the present line of mice onto genetically modified strains, one can also study the role of specific genes in type II cell function. Clearly, the use of EGFP as a noninvasive marker will enhance our ability to study type II cells under a variety of experimental conditions.
In summary, we have shown that mouse type II cells can be identified and isolated on the basis of transgenic expression of EGFP. As we identify new cell type-specific promoters and develop new isoforms of GFP that fluoresce at different wavelengths, our ability to track multiple cell types at the same time should increase. Indeed, the present method for isolating EGFP-expressing type II cells should be amenable to isolating other cell types as new lines of intrinsically fluorescent mice become available. Until that time, the present isolation procedures used with this line of mice provides an exciting new opportunity to investigate mouse type II epithelial cells.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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