Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948
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ABSTRACT |
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Tracheal, renal, salivary, and pancreatic epithelial cells from
cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) /
] and non-CF mice that carry a
temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were
isolated and maintained in culture under permissive conditions (33°C
with interferon-
). The resultant cell lines have been in culture for
>1 year and 50 passages. Each of the eight cell lines form polarized
epithelial barriers and exhibit regulated, electrogenic ion transport.
The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl
permeability and cAMP-stimulated
Cl
secretion. In contrast, the four CFTR
/
cell lines
(mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated
Cl
secretory responses. Ca2+-activated
Cl
secretion is retained in both CF and non-CF cell
lines. Thus we have generated genetically well-matched epithelial cell
lines from several tissues relevant to cystic fibrosis that either
completely lack CFTR or express endogenous levels of CFTR. These cell
lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.
cystic fibrosis; murine epithelial cell lines; ts-sv40 large T antigen; cystic fibrosis transmembrane conductance regulator; calcium-activated chloride secretion
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INTRODUCTION |
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MUTATIONS in the
cystic fibrosis transmembrane conductance regulator (CFTR) are
responsible for cystic fibrosis (CF), and the primary defect in CF is
the loss of cAMP-regulated anion conductance in the apical plasma
membrane of epithelial cells in affected tissues (29, 33).
Identification, cloning (34, 35), and heterologous
expression of CFTR and mutant forms of CFTR provide direct evidence
that the protein functions as a cAMP-regulated anion channel (2,
3). It is not certain how loss of apical membrane
Cl conductance alone leads to the wide spectrum of
phenotypic abnormalities associated with defective CFTR
(37), including aberrant regulation of Na+
channels (9, 10, 41) and non-CFTR Cl
channels (38), altered regulation of exocytosis and
endocytosis (13), abnormal composition of macromolecules,
and increased adherence of certain bacteria to airway epithelial cells
(12). A number of mechanisms have been proposed to explain
CF pathophysiology (4, 38, 39); however, unifying
hypotheses are few, and the relevance of certain observations to native
epithelial cell function is unclear. Furthermore, most studies of CFTR
function have been performed using airway and intestinal (T84)
epithelial cells (7, 8, 43) or heterologous expression
systems (5). Epithelial cells from other tissues affected
by CF have received relatively little attention, primarily due to lack
of access to appropriate human biological material (6).
The development of the CF mouse has expanded the number of epithelial
cell types available for study (16, 21, 40).
Primary culture of human epithelial cells (9) and
immortalized epithelial cell lines (24, 25, 27, 28, 45,
46) have been widely used in biomedical research and have proven
particularly useful for studies of CF and CFTR. Many of the
immortalized CF and non-CF epithelial cell lines were generated by in
vitro transfection of human airway epithelial cells with either SV40
large T antigen (24, 27, 28, 46) or human papilloma virus
(45). The degree of differentiation appears to vary
widely, as many of the cell lines do not form functional epithelial
tight junctions (24, 46). However, the CF phenotype,
namely loss of cAMP-stimulated Cl permeability, is
retained (24, 27, 28, 45, 46). Immortalized cell lines
have also been developed from genetically modified mice that carry an
SV40 large T antigen transgene (25). Recently, Jat and
coworkers (26) produced a transgenic mouse line
(H-2Kb-tsA58; ImmortoMouse) that carries a
thermolabile mutant of SV40 large T antigen under the control of a
ubiquitous interferon-
(IFN-
)-inducible promoter. This mouse line
has been used to generate several conditionally immortalized cell lines
to date (14, 26, 32, 42, 44). The major theoretical
advantages of cell lines generated in this way are 1) the
avoidance of unpredictable characteristics of in vitro transfection
with oncogenes (e.g., variable copy number and multiple sites of
integration), 2) the ability to immortalize a heterogeneous
population of cells from an epithelium (e.g., ciliated, goblet, and
basal cells from the airway) from which clones with specific properties
may be selected at a later time, and 3) the opportunity to
control large T antigen levels and thereby promote differentiation by
switching the cells from permissive to nonpermissive culture conditions
(36). The goal of this work was to cross the ImmortoMouse
(26) with the University of North Carolina (UNC)
CF knockout mouse (CFTR S489X) (40) and develop genetically well-matched, conditionally immortalized CF and non-CF epithelial cell lines.
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METHODS |
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Animals
Male mice, homozygous for a temperature-sensitive SV40 large T antigen transgene (ImmortoMouse; CBA/ca X C57B1/10 strain; Charles River Laboratories) (26), were bred with female mice that were heterozygous for the S489X CFTR mutation (UNC; CFTR +/Tissue and Cell Isolation
Collecting tubule cell lines.
Ten mice that carried at least one copy of the
H-2Kb-tsA58 transgene and that were heterozygous
for the CFTR allele were killed, and the kidneys were removed, sliced,
and digested with collagenase type IV (0.5% wt/vol) in Hanks'
balanced salt solution (HBSS) for 30 min at 37°C. The digest was
centrifuged (800 g for 5 min), and the cell pellet was
resuspended in cold culture medium with 10% fetal bovine serum (FBS),
centrifuged again, and resuspended in 5 mM glucose in
phosphate-buffered saline (PBS). The cell suspension was plated onto
tissue culture dishes (Falcon 1058; Falcon-Becton Dickinson, Lincoln
Park, NJ) coated with Dolichos biflorus agglutinin (DBA;
4°C, 10 µg/ml in 0.1 M NaHCO3). DBA has been shown to
specifically label the collecting duct (principal and intercalated
cells) of the mouse kidney (30). Cells were allowed to
adhere for 45 min at 4°C. Unbound cells were removed by being washed
three times with PBS-glucose at 4°C. Lectin-adhered cells were eluted
by being incubated in 10 ml of 150 mM galactose in PBS for 5 min. Cells were washed in PBS-glucose, centrifuged (800 g for 5 min),
washed in culture medium, centrifuged (800 g for 5 min),
resuspended, and plated in culture media at a density of 2 × 105 cells/ml. Cells were maintained as primary cultures at
37°C for 7 days in defined basal media for collecting tubule
epithelial cells (CT media). Once colonies were established,
recombinant mouse IFN- (10 U/ml) was added to the basal CT media,
and cultures were expanded at the 33°C permissive temperature. The
resulting cell line is identified as mCT1 (42). An
identical procedure was used to isolate CT cells from six mice that
carried at least one copy of the H-2Kb-tsA58
transgene with both CFTR alleles disrupted. The resulting cell
line is identified as mCT1-CF.
Pancreas cell lines. Four mice that carried at least one copy of the H-2Kb-tsA58 transgene and were heterozygous for the CFTR allele were killed, and the pancreati were removed. The tissues were placed in a HEPES-buffered Ringer solution (HR) that contained 0.25 mg/ml collagenase type I, 0.25 mg/ml collagenase type IV, and 0.1 mg/ml soy bean trypsin inhibitor. The tissues were minced with scissors and digested for 45 min at 37°C. At 15-min intervals, the tissue fragments were disrupted by repeat passage through a plastic pipette. The resulting tissue digest was passed through a nylon filter (149 × 149 µm), and the material trapped by the filter was retained for culture. The tissue fragments were resuspended in exocrine media and plated on tissue culture dishes. A combination of serum-free media and differential trypsinization was used to eliminate fibroblast contamination. The resulting cell line is identified as mPEC1. An identical procedure was used to isolate pancreatic epithelial cells from four mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mPEC1-CF.
Salivary gland cell lines. Three mice that carried at least one copy of the H-2Kb-tsA58 transgene and were homozygous for the wild-type CFTR allele were killed, and the submandibular salivary glands were removed. The tissues were placed in HR that contained 0.25 mg/ml collagenase type I, 0.25 mg/ml collagenase type IV, and 0.1 mg/ml soy bean trypsin inhibitor. The tissues were minced with scissors and digested for 60 min at 37°C. At 15-min intervals, the tissue fragments were disrupted by repeat passage through a plastic pipette. At the end of this period, the cell suspension was layered over a 4% bovine serum albumin (BSA in HR) solution and was allowed to settle for 10 min on ice. The supernatant was removed, and the resulting pellet was resuspended in exocrine media and plated in tissue culture dishes. After several passages, the contaminating fibroblasts were removed by differential trypsinization. The resulting cell line is identified as mSEC1. An identical procedure was used to isolate salivary gland epithelial cells from three mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mSEC1-CF.
Tracheal epithelial cell lines. Three mice that carried at least one copy of the H-2Kb-tsA58 transgene and were heterozygous for the CFTR allele were killed, and tracheas were removed. Isolated trachea were cleaned of connective tissue, opened along the posterior surface, and pinned to expose the epithelial surface. The tissues were exposed to 0.1% protease (type XIV, Sigma) and 0.1% collagenase (type IV, Sigma) in Ca2+- and Mg2+-free HBSS that contained 5 mM EDTA at 37°C for 20 min. The epithelium was freed from tracheal rings and lamina propria by pipetting a steady, forceful stream of fluid over the partially digested tracheal tissue. Tracheal epithelial cells were rinsed, centrifuged, resuspended in small airways growth media (SAGM; Clonetics), and plated on vitrogen gel-coated six-well culture plates. Cells were grown on vitrogen gels for three passages. Tracheal cell cultures were subsequently expanded and maintained on tissue culture dishes. The resulting cell line is identified as mTEC1. An identical procedure was used to isolate tracheal epithelial cells from two mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mTEC1-CF.
Cell culture.
Renal cell lines were maintained in culture media (CT media) that
contained a 1:1 mix of Dulbecco's modified Eagle's medium (DMEM) and
Ham's F-12 medium supplemented with 1.3 µg/l sodium selenite, 1.3 µg/l 3,5,3'-triiodo-L-thyronine, 5 mg/l insulin, 5 mg/l transferrin, 25 µg/l prostaglandin E1, 2.5 mM
glutamine, 5 nM dexamethasone, 50,000 U/l nystatin, 50 mg/l
streptomycin, 30 mg/l penicillin G, and 10,000 U/l recombinant mouse
IFN-. mCT1 and mCT1-CF cells were maintained on plastic tissue
culture dishes in CT media in a humidified 33°C incubator with 5%
CO2. Media was changed every other day, and cells were
passaged weekly. Cells used for experiments reported here were between
passages 15 and 25. Pancreatic and salivary gland
epithelial cell lines were maintained in culture media (exocrine media)
that contained 1:1 mix of DMEM and Ham's F-12 medium supplemented with
0.5 mM isobutyl methyl xanthine, 2 mM glutamine, 10 µg/l epidermal
growth factor, 100 mg/l streptomycin sulfate, 60 mg/l penicillin G,
50,000 U/l nystatin, 2.5% FBS, and 10,000 U/l IFN-
. Salivary and
pancreatic epithelial cells were maintained on plastic tissue culture
dishes in exocrine media in a humidified 33°C incubator with 5%
CO2. Media was changed every other day, and cells were
passaged weekly. Cells used for experiments reported here were between
passages 10 and 25. Tracheal epithelial cells were
maintained in SAGM (Clonetics) supplemented with 500 mg/l BSA, 0.5 µg/l human epidermal growth factor, 5 mg/l insulin, 6.5 µg/l 3,5,3'-triiodo-L-thyronine, 10 mg/l transferrin, 30 mg/l bovine pituitary extract, 0.5 mg/l epinephrine, 0.5 mg/l
hydrocortisone, 0.1 µg/l retinoic acid, 50 mg/l gentamycin sulfate,
50 µg/l amphotericin B, and 10,000 U/l IFN-
. Tracheal epithelial
cells were maintained on plastic tissue culture dishes in SAGM in a
humidified 33°C incubator with 5% CO2. Media was changed
every other day, and cells were passaged weekly. Cells used for
experiments reported here were between passages 15 and 30.
Immunolocalization of ZO-1. Cells were seeded onto Costar Transwell clear polyester filters and maintained under permissive conditions until the cultures became confluent. The epithelial monolayers were rinsed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. The monolayers were then washed three times with PBS, permeabilized by exposure to 0.1% Triton X-100 in PBS for 5 min, and then washed three times with PBS. The monolayers were blocked with 10% FBS in PBS and then incubated with primary antibody (diluted 1:10; ZO-1; R26.4C; obtained from the Developmental Studies Hybridoma Bank at the Univ. of Iowa) for 60 min at room temperature. The cells were washed three times with PBS and then exposed to secondary antibody (diluted 1:100; FITC-conjugated Affinipure goat anti-rat IgG; Jackson Immunochemicals) for 60 min at room temperature. The monolayers were washed three times with PBS, and a section of the filter was cut, mounted on a glass slide with a drop of Slow Fade (Molecular Probes), covered with a coverslip, and sealed with clear nail polish. The samples were examined using a confocal microscope.
Transepithelial electrical measurements.
Cells were seeded (1-3 × 105 cells/filter) on
collagen-coated, permeable supports (Millicell-CM 12 filters,
Millipore) cut to a height of 4 mm, with the "feet" removed
(17). The filter surface was coated with 125 µl/cm2 calf skin collagen (Sigma) dissolved in acetic
acid (7.5 mg/ml 0.2% glacial acetic acid) and allowed to dry. The
collagen coating was cross-linked by exposure to ammonium hydroxide
vapors (3.5% solution) for 10 min followed by immersion in
glutaraldehyde (2.5%) for 10 min. This procedure was followed by a
thorough rinsing in distilled water, 70% ethanol, distilled water, and
finally, culture media. Filter-grown cells were cultured with IFN-
at 33°C for 7-14 days. Media was changed every 48 h. Cell
monolayers grown on modified supports were clamped between Lucite flux
chambers and bathed on both sides by equal volumes (usually 6-10
ml) of Krebs-Ringer bicarbonate (KRB) solution. The solutions were
circulated through the water-jacketed glass reservoir by gas lifts
(95% O2-5% CO2) to maintain solution
temperature at 37°C and pH at 7.4. Transepithelial voltage
difference (VT) was measured between two
Ringer-agar bridges, each positioned within 3 mm of the monolayer
surface. Calomel half-cells connected the bridges to a high-impedance
voltmeter. Current from an external direct current source was passed by
silver-silver chloride electrodes and Ringer-agar bridges to clamp the
spontaneous VT to 0. The current required
(short-circuit current, Isc) was corrected for
solution and filter series resistance. Monolayers were maintained under
short-circuit conditions except for brief 3- to 5-s intervals when the
current necessary to clamp the voltage to a nonzero value (usually
+2 mV) was measured to calculate transepithelial resistance
(RT).
36Cl efflux.
Efflux assays were performed as described previously (1,
31). Briefly, cells were grown to confluence in 35-mm tissue culture dishes. The monolayers were then washed three times with HR to
remove media, and monolayers were incubated with
36Cl
(NaCl, 5 µCi/ml; Amersham, Arlington
Heights, IL) in 1 ml HR for 1 h. After the cells were loaded with
36Cl
, they were rapidly washed (3 times with
warmed isotope-free HR) to remove extracellular
36Cl
. Efflux of
36Cl
was measured at 30-s intervals for 8 min. The effect of elevation of cAMP on 36Cl
efflux was determined by adding forskolin (10 µM) and isobutyl methyl
xanthine (100 µM) during time intervals of 2 to 8 min. After the last
sample was removed, the cells were lysed by the addition of 0.5 ml of 1 N HCl for 20 min. The sample was neutralized with NaOH. All samples
were mixed with liquid scintillation fluid (Ecolume, ICN) and assayed
for 36Cl
activity (LS 5801; Beckman
Instruments, Fullerton, CA). The apparent rate constant (r,
in min
1) was calculated for each efflux interval from the
following equation: r = [ln(C1)
ln(C2)]/(t2
t1), where ln(C1) and
ln(C2) are the natural logs of the percentage of counts
remaining in the cell layer, at times t1 and
t2, respectively.
Solutions and chemicals. HR was composed of (in mM) 10 HEPES, 138 NaCl, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose. KRB was composed of (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose.
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RESULTS |
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Epithelial cell lines were successfully derived from trachea,
pancreas, salivary gland, and renal CT from CF and non-CF mice that
carried the temperature-sensitive SV40 transgene. Each of the eight
cell lines had been maintained in culture under permissive conditions
(33°C plus INF-) for >1 yr with >50 passages. Multiple attempts
to generate cell lines from the small intestine and colon were not
successful. The cell lines grow as epithelial monolayers, and each cell
line expresses the epithelial tight junction protein ZO-1
(Fig. 1), consistent with the formation
of functional tight junctions (as revealed by measurements of
transepithelial electrical resistance).
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Cell Line Genotypes
The genotypes of the cell lines were determined by PCR analysis of genomic DNA, and the results are shown in Fig. 2. The CF cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) were negative for the wild-type CFTR allele (faint 200-bp bands are non-CFTR PCR products) and positive for the S489X neodisrupted allele of CFTR. In contrast, three of the non-CF cell lines (mTEC1, mCT1, and mPEC1) carried both the wild-type CFTR allele and the S489X neodisrupted allele, whereas one of the non-CF cell lines (mSEC1) was positive for the wild-type CFTR allele and negative for the S489X neodisrupted allele of CFTR. All eight cell lines were positive for the SV40 transgene. The results were as expected based on the genotypes of the animals used for cell isolation.
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cAMP-Regulated Cl Permeability
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Electrogenic Ion Transport
Transepithelial electrical measurements were made on epithelial monolayers to determine whether the cell lines formed polarized epithelial barriers and expressed the appropriate Cl
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Effects of Nonpermissive Culture Conditions on Ion Transport Properties of mCT1 Cells
The cell lines described in this report were derived from animals that carry a temperature-sensitive form of large T antigen. We have previously shown that the amount of large T antigen and the rate of cell proliferation are reduced by placing the cultures at 39°C and removing the INF-
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DISCUSSION |
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The purpose of this work was to generate genetically well-matched,
immortalized epithelial cell lines from CF and non-CF mice. The results
of the 36Cl efflux studies demonstrate that
the non-CF cell lines each respond to elevation of cAMP with a
characteristic increase in Cl
permeability. The small
increase in 36Cl
efflux seen in mSEC1-CF and
mPEC1-CF cells (after a 3-min delay) suggests that in these cell types,
elevation of cAMP stimulates bumetanide-sensitive
Na+-K+-2Cl
cotransport. Without
concurrent stimulation of apical Cl
conductance,
activation of basolateral
Na+-K+-2Cl
cotransport would not
be expected to increase Isc (see Fig. 4). The
transepithelial electrical measurements provide direct evidence for
cAMP-activated Cl
secretion in non-CF cell lines but not
in the corresponding CF cell lines. Therefore, as expected, the
cAMP-dependent Cl
secretory phenotype of the cell lines
accurately reflects the genotypes of the animals from which the cells
were derived (9, 11, 28).
A variety of secondary defects have been identified in CF cells,
including hyperabsorption of Na+ (9, 20, 23)
and altered cAMP-dependent regulation of epithelial Na+
channels (10, 37, 41) and outward-rectifying
Cl channels (38). Six of the eight cell
lines that we generated exhibited small, amiloride-sensitive currents;
however, Na+ hyperabsorption was not observed in the CF
cell lines. This is not unexpected, since freshly isolated CF mouse
trachea does not exhibit Na+ hyperabsorption compared with
non-CF trachea (22). The reason for tissue- and
species-specific Na+ hyperabsorption (9, 20,
23) in CF is not known but may depend on the relative and
absolute levels of expression of CFTR and epithelial Na+
channel. CFTR is also known to regulate cAMP-activated, DIDS-sensitive Cl
channels, perhaps via release of ATP, although this
hypothesis remains controversial (37, 38). CFTR-dependent
activation of DIDS-sensitive Cl
secretion is observed in
mPEC1 monolayers but not in mPEC1-CF cells (data not shown). Thus
murine pancreatic cell lines appear to retain abnormal regulation of
DIDS-sensitive Cl
channels and may be useful for studies
of the interaction of CFTR and non-CFTR Cl
channels.
It is difficult to make comparisons between the cell lines that we
generated and native murine epithelia due to the paucity of ion
transport data from freshly isolated tissues. Transepithelial ion
transport data are not available from native murine pancreatic ducts;
however, Githens et al. (19) reported that primary
cultures of mouse pancreatic ducts expressed amiloride-sensitive
Na+ absorption and cAMP- and Ca2+-stimulated
Cl secretory responses. The responses of the mPEC1 cell
line resembled those reported by Githens et al. (19), but
quantitative comparisons cannot be made since they showed only single
traces with no summary data. We are unaware of transepithelial ion
transport data from murine salivary ductal epithelial cells. A number
of renal epithelial cell lines have been generated from various nephron
segments, including CT (36, 42). Nearly all of the CT cell
lines express amiloride-sensitive Na+ absorption and
cAMP-stimulated Cl
secretion, similar to the results
obtained with the mCT1 cell line. The relatively small
amiloride-sensitive Isc of mCT1-CF cells was
unexpected but is probably unrelated to the lack of CFTR expression,
since amiloride-sensitive Na+ absorption appears to be
poorly retained in epithelial cell lines. We have previously shown that
mCT1 cells express aquaporin-2 and vasopressin receptors
(42), properties characteristic of CT principal cells. The
ion transport properties of freshly isolated tissues and primary
cultures of murine tracheal epithelium have been established. Grubb and
coworkers (22) reported cAMP-activated Cl
secretion of ~10 µA/cm2 in both non-CF and CF mouse
trachea, whereas primary cultures of non-CF and CF tracheal
epithelium responded to elevation of cAMP with ~5 and 0 µA/cm2, respectively (15). The anomalous
secretory response of CF trachea to cAMP is thought to be mediated by
cAMP-dependent release of Ca2+ and activation of
Ca2+-stimulated Cl
secretion
(22). The response is absent in primary cultures of CF
tracheal cells (15) and in the immortalized mTEC1-CF cell line (this report). The cAMP-activated secretory response of mTEC1 cells might be enhanced by modifications in culture conditions such as
addition of cholera toxin to the media and/or growth at an air-liquid
interface. The Cl
secretory response elicited by
elevation of intracellular Ca2+ in CF and non-CF trachea
(~25-30 µA/cm2) (15) and primary
cultures of non-CF and CF tracheal cells (~20-40
µA/cm2) (20) is similar to that observed in
our immortalized tracheal cell lines (~17-27
µA/cm2) (this report).
The strategy used to generate the cell lines (CF knockout mice crossed
with the ImmortoMouse) avoided the problems (variable integration site
and transgene copy number) associated with in vitro immortalization of
primary cell cultures. Furthermore, the use of a temperature-sensitive
SV40 large T antigen provides an opportunity to control large T antigen
levels and cell proliferation. The results presented above were
obtained from cells maintained under permissive growth conditions
(33°C with IFN-); however, several cell lines derived from the
ImmortoMouse show tissue-specific differentiation when the cells are
placed under nonpermissive growth conditions (14, 26, 42,
44). The mCT1 cells are derived from principal cells of the CT
and are expected to exhibit amiloride-sensitive Na+
absorption. Thus the increase in amiloride-sensitive Na+
absorption coupled with the decrease in cAMP-stimulated
Cl
secretion (Fig. 5) when the cells are maintained under
nonpermissive conditions is consistent with acquisition of a more
differentiated transport phenotype. Additional studies will be required
to examine the effect of nonpermissive growth conditions on each of the
cell lines.
The cell lines that we have generated are unique in several regards:
1) they represent the first cell lines obtained from CF
knockout mice, 2) the lines are derived from several tissues relevant to CF, 3) the lines are genetically well matched,
and 4) the use of a temperature-sensitive SV40 large T
antigen oncogene should provide a means to regulate growth and
differentiation in culture. The cell lines should prove useful for
studies of the role of CFTR in normal epithelial cell physiology. Since
the CF cells are derived from CFTR /
mice, they represent a null background suitable for studies of mutant forms of CFTR and should help
to define alterations in cell function associated with loss of CFTR
and/or specific CFTR mutations in the context of an epithelial cell.
Finally, the non-CF cell lines will be useful as model systems for
studies of regulation of epithelial function.
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ACKNOWLEDGEMENTS |
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We thank Mike Haley for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-53318, the Cystic Fibrosis Foundation, and the Polycystic Kidney Research Foundation.
Address for reprint requests and other correspondence: C. U. Cotton, 2109 Adelbert Rd., Biomedical Research Bldg., Cleveland, OH 44106-4948 (E-mail: cuc{at}po.cwru.edu).
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.
Received 22 May 2000; accepted in final form 29 August 2000.
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