Key words: CFTR; cell culture; gallbladder; cystic fibrosis; epithelium; murine.. In parallel experiments, cells were plated onto Vi- trogen-coated 35-mm plastic plates and fed with cond
Trang 1In Vitro Cell Dev Biol. Anilna133:104-109 February 1997
© 1997 Society for In Vitro Biology
1071-2690/97 $05.00 + 0.00
ISOLATION A N D LONG-TERM CULTURE OF GALLBLADDER EPITHELIAL CELLS
FROM WILD-TYPE AND CF MICE
RAHUL KUVER ~ CHRISTOPHER SAVARD, TOAN D NGUYEN, WILLIAM R A OSBORNE AND SUM P LEE
Departments of Medicine (R K., C S T D N., S P L ), and Pediatrics (W R A 0 ), University of Washington and VA Medical Center,
Seattle, Washington 98195
(Received 8 November 1995; accepted 4 March 1996)
SUMMARY
Mice with targeted disruption of the cfir gene show pathophysiologic changes in the gallbladder, which correlate with
hepatobiliary disease seen in cystic fibrosis patients As gallbladder epithelium secretes mucin, and as this epithelium
consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the
complex cellular functions of this protein The size and anatomic location of the murine gallbladder makes such studies
difficult in vivo Therefore, the need exists for in vitro models of gallbladder epithelium We describe a method to isolate
and culture murine gallbladder epithelium from wild-type and CF mice Ceils were grown in a monolayer on porous inserts
over a feeder layer of fibroblasts These nontransformed cells can be successively passaged and maintain a well-differen-
tiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with
prominent microvilli and intercellular junctions Organotypic cultures showed columnar cells simulating in vivo morphology
This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium
Key words: CFTR; cell culture; gallbladder; cystic fibrosis; epithelium; murine
INTRODU CT1ON Mouse models of cystic fibrosis have been created by targeted
disruption of the cfir gene (7,11,31,39) These models have been
valuable for delineating CFTR-related organ pathology In one such
model, created at the University of North Carolina (UNC), homozy-
gous cfir ( - / - ) mice showed significant pathology related to the
intestinal tract, with death occurring in the postnatal period from
complications relating to intestinal blockage in a syndrome resem-
bling meconium ileus (40) The trachea (15), nasal epithelium (14),
jejunum (13), oviduct (24), colon (8), small intestine (42), pancreatic
acinar cells (10), and pancreatic duct epithelium (12) have been
studied either in vivo or in cell culture in this and other mouse
models These studies have provided a detailed understanding of the
electrophysiologic role of CFTR in these cell types
CFTR possesses diverse cellular functions (32) Negative regula-
tion of an epithelial sodium channel (41), regulation of the outwardly
rectifying chloride channel via transport of ATP (38), and its origi-
nally demonstrated role as a cAMP-regulated apical membrane chlo-
ride channel (36) point to the complex nature of this molecule In-
tracellular CFTR functions involving the biosynthetic pathway
(5,22), endosomal acidification (3), and membrane recycling (4) are
under active investigation
A central question is the relationship between defective or absent
CFTR and the hyperviscous, abundant mucus found in various organ
ductal systems The gallbladder is an ideal organ for studying the
~To whom correspondence should be addressed at Division of Gastroen-
terology, Box 356424, University of Washington, 1959 NE Pacific Street,
Seattle, Washington 98195
pleiotropic cellular roles of CFTR The gallbladder is abnormal in
CF patients, showing hypoplasia, thick mucus, and calculi (1) In UNC mice dying from intestinal blockage in the early postnatal pe- riod, the gallbladder was often ruptured when examined postmortem
(39) Even in apparently healthy cfir ( - / - ) mice, polymorphonu- clear cell infiltration throughout the gallbladder wall suggested on-
going inflammation (39) In vivo electrophysiologic studies are diffi-
cult to perform on the nmrine gallbladder, however, due to its size and location Therefore, the need exists to culture murine gallbladder epithelium in order to advance these studies
MATERIALS AND METHODS
Materials Chemicals of analytical grade were obtained from Sigma Chem- ical Co (St Louis, MO) except where noted Vitrogen, a bovine dermal col- lagen, was purchased from Celtrix Labs (Palo Alto, CA) Falcon culture plates were from Becton Dickinson (Franklin Lakes, N J) and the Transwell inserts (24-mm diameter, 3-p.m pore size) were obtained from Costar (Cambridge, MA)
Cell isolation and culture Mice with targeted disruption of the cftr gene
created at the University of North Carolina (39) were maintained at the Uni-
versity of Washington Animal Care Facility For the initial isolation, 2 cfir ( - / - ), 6 cfir heterozygous ( + / - ) and 2 wild-type cfir ( + / + ) littermates
were used The isolation procedure was repeated 6 months later with another cohort of mice, with identical results Mice were more than 20 d old Mice were killed by cervical dislocation and washed with 90% ETOH, and a lap- arotomy was performed under sterile conditions The gallbladder was ex- posed, removed from the common bile duct, and punctured, and bile was aspirated Each gallbladder was cut longitudinally, then washed with phos- phate-buffered saline (PBS) multiple times Five milliliters of 0.25% trypsin/ 0.1% EDTA was added, followed by incubation for 20 min at 37 ° C with frequent vortexing Ten milliliters of Eagle's minimum essential medium con- taining 10% fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin/ml,
104
Trang 2inserts was between 60 000 and 250 000 cells per well When confluent, each well contained approximately 3 million cells The insert allowed sepa- rate access to both the apical and basolateral sides of a confluent monolayer For cryopreservation, the epithelial cells were frozen in medium containing 10% dimethyl sulfoxide In parallel experiments, cells were plated onto Vi- trogen-coated 35-mm plastic plates and fed with conditioned medium ob- tained from confluent monolayers of human gallbladder myofibroblasts and mixed 1:1 with MEM
Organotypic cultures This culture method permits greater morphologic dif- ferentiation of ceils, allowing comparison to in vivo nmrphologic character- istics (26) The culture method features an air-liquid interface, with the apical
FIG 1 Phase-contrast light micrograph of mouse gallbladder epithelial
cells in monolayer culture Shown is a subconfluent monolayer of cfir ( + / + )
ceils grown on Vitrogen with human gallbladder myofibrublast-conditioned
media Original magnification )< 20
100 p,g streptomycin/ml, 20 mM HEPES, 1.0 g glucose/1, supplemented with
5 ~tg insulin/ml from bovine pancreas, 5 p,g human transferrirdml, 5 ng so-
dium selenite/ml {ITS supplement), 1 X vitamins solution, and 1 X nones-
sential amino acid solution (Sigma) was then added This medium will sub-
sequently be referred to as MEM Trypsin was inactivated by the fetal bovine
serum The samples were transferred to a tube and centrifuged, and the su-
peruatant was discarded The pellet was resuspended in 2 ml of medium and
pipetted onto a 35-mm plate Many sheets of cells were visible which were
separate from the gallbladder wall fragments These gallbladder wall frag-
ments were manually removed with forceps, and the medium with suspended
cells was pipetted onto Vitrogen-coated inserts above a confluent layer of
human gallbladder myofibroblasts The seeding density of the gallbladder
epithelial cells was approximately 1 X 105 per insert One insert was plated
out from each gallbladder The remaining cells in the 35-mm plate were more
than 95% viable by Trypan blue exclusion MEM was added into the apical
and basolateral compartments (2 ml each side) and the inserts were placed
in a 37 ° C humidified incubator (5% CO2/95% air) Twenty-four hours after
plating, the unattached epithelial cells were removed during replacement of
the medium, and the attached ceils became confluent 6 d after plating
The myofibroblast cell line was isolated from a normal human gallbladder
after a 1-h treatment with 0.5% trypsin/0.2% EDTA, which enabled isolation
of both epithelial cells and myofibroblasts Myofibroblasts preferentially at-
tached to and grew on plastic culture plates, whereas gallbladder epithelial
cells did not Therefore, gallbladder myofibroblasts were cultured on 60-ram
plastic culture plates in MEM The seeding density was approximately '1 X
105 cells per 60-mm culture dish The MEM was replaced every 2 d until
cells were confluent which required approximately 1 week Once confluent,
these cells were trypsinized with 0.25% trypsin/0.1% EDTA; then MEM was
added, and the ceils were plated onto 35-mm six-well plates at a seeding
density of 1 x 105 cells per well Confluency was achieved in approximately
1 week, at which point the cells stopped dividing and could be maintained
in culture for up to 2 weeks Vitrogen-coated inserts containing mouse gall-
bladder epithelial cells were placed above these wells, as described earlier
The mouse gallbladder epithelial cells and the human gallbladder myofi-
broblasts were fed twice weekly (2 ml apical/2 ml basolateral compartments)
with Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, MD)
with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin/ml, 100
~tg streptomycin/ml, 1 mM sodium pyruvate, 4.9 g glucose/1 supplemented
with 5 ~tg insulin/ml from bovine pancreas, 5 ~g human transferrin/ml, 5 ng
sodium selenite (ITS supplement)/ml, 1 X nonessential amino acids solution
and 1 X vitamins solution (Sigma), hereafter referred to as DMEM Whereas
DMEM was used from Passage 3 onwards, the cells could be maintained
beyond Passage 3 in MEM Care was taken during feeding to not contaminate
the epithelial cells in the upper compartment of the insert with the myofi-
broblasts from the bottom compartment The cells were passaged when they
became confluent, usually 6-10 d after plating, with 0.25% trypsin/0.1%
EDTA to release the cells When being passaged, the seeding density for new
A
B
FiG 2 Electron micrographs of (A) cfir (+ / +) and (B) cfir ( - / - ) mouse gallbladder epithelial cells grown on Vitrogen-coated inserts over a feeder layer of human gallbladder myofibroblasts Both cell types are at Passage 3 Apical microvilli, lobed nuclei, mitochondria, and apically localized secre- tory granules are evident Original magnifications: (A) X 8000; (B) X 21 000
Trang 3106 KUVER ET AL
FIG 3 Light micrograph of cfir ( + / + ) mouse gallbladder cells in or-
ganotypic culture The cells are at Passage 5 Tall, columnar cells with basally
localized nuclei are evident Original magnification × 40
surface of cells facing the air and the basolateral surface bathed in medium
It is not a method for maintaining cells in long-term cuhure Full details of
the technique is provided elsewhere and was performed as described (26)
In brief, mouse gallbladder epithelial cells, maintained on inserts as de-
scribed earlier, were trypsinized with 0.25% trypsin/0.1% EDTA, resus-
pended in medium, and plated at a seeding density of 5 × 10 ~ cells onto a
freshly prepared matrix consisting of seven parts rat-tail collagen Type 1
(Collaborative Biomedical Products, Bedford MA) at 3 mg/ml, and one part
fetal bovine serum containing human neonatal foreskin fibroblasts (26) The
collagen-fibroblast matrix was preincubated at 37 ° C for 15 min in order to
solidify After 2 h to allow epithelial cel] attachment to the collagen matrix,
the matrix was gently scraped from the sides and bottom of the dish to allow
the collagen to contract evenly This resulted in a matrix approximately i cm
in diameter and 1.5 mm thick The ceils were submerged in medium (26) and fed daily After 5 to 7 d, they were transferred to metal grids placed on 60-mm organ tissue culture plates with culture medium in the center well
In this system, the epithelial cells were exposed to 5% CO2/95% air on the apical surface and received all their nutritional support vectorally from the basolateral side as the medium diffused through the collagen-fibroblast layer After 5 d, the cells were fixed in Hollande's or half-strength Karnovsky's fixative for thin-section light microscopy or electron microscopy, respectively
Transmission electron microscopy Confluent cells were washed with PBS and fixed in half-strength Karnovsky's reagent The cells were postfixed in 2% osmium tetroxide with 0.1 M cacodylate buffer, dehydrated in graded ethanols, and embedded in Epon (Pella Inc., Redding, CA) Thin sections were stained with uranyl acetate and lead tartrate and photographed in a Philips EM410 transmission electron microscope
Flow cytor, vetry Cells were plated at 1 X 105 cells per well The culture medium was changed every 2 d At Days 2, 4, 9, and 14 after plating, du- plicate wells were washed and trypsinized Staining and flow analysis were done as described (35) Cell pellets were resuspended in 1 ml of 4',6-diam- idino-2-phenylindole-staining solution with 10% dimethyl sulfoxide (vol/vol) The samples were evenly mixed and stored frozen at - 70 ° C After thawing, the stained cell suspension was analyzed with an epi-illumination flow system designed by GOHDE (ICP22A; Phywe AG Gottingen, Germany, now Ortho Diagnostic Systems, Westwood, MA)
Western blotting Cells were harvested with 0.25% trypsin/0.1% EDTA, washed, pelleted, and solubilized in 0.125 M Tris-HC1, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and protease inhibitors Proteins (200 ~g/lane) were then separated by SDS-polyacrylamide gel elec- trophoresis and blotted onto polyvinylidene membrane (Immobilon-P, Milli- pore Corp., Bedford, MA) CFTR was detected with sequential 1-h incuba- tions with a 1:2000 dilution of a polyclonal CFTR antibody, a 1:40 000 dilution of biotin-goat-anti-rabbit antibody (Vector Labs, Burlingame, CA), and streptavidin-horseradish peroxidase Peroxidase activity was demon- strated with enhanced chemiluminescence (Amersham, Arlington Heights, IL) The CFTR polyclonal antibody was raised in rabbits against the 13 amino-acid C-terminal sequence of human CFTR (22)
FIG 4 Electron micrographs of (A) cfir ( + / + ) and (B) cfir ( - / - ) mouse gallbladder ceils in organotypic culture Both cell types
are at Passage 5 A higher density of apical microvilli when compared to cells grown on inserts, and numerous apical secretory granules
are evident Original magnifications: (A) X 9000; (B) X 16 000
Trang 4Measurement of monolayer resistance [29] Wild-type mouse gallbladder
epithelial cells were cultured on Vitrogen-coated inserts as described earlier
and monolayer eonfluenee was confirmed with an inverted microscope The
membrane supporting the cell monolayer was then excised and mounted in
a modified Ussing chamber with a circular aperture of 0.95 cm 2 Both sides
of the monolayer were bathed in Ringer's solution (115 mM NaC1, 3L.2 mM
CaC12, 1.2 mM MgCI2, 0.4 mM KHzPO ~, 2.5 mM K2HPO4, 25 mM HCO3, and
10 mM glucose) warmed to 37 ° C with a circulating water jacket and gently
mixed and aerated with a constant inflow of 95% air/5% CO 2 The two com-
partments of the Ussing chamber, corresponding to the apical and basolateral
surfaces of the monolayer, were next connected to an automatic voltage clamp
(model DVC-1000, WPI, Sarasota, FL) through agar bridges and Ag-AgC12
electrodes
After equilibration, a constant current of 100 pA was circulated across the
mooolayer and the resulting potential difference between the two compart-
ments recorded The resistance of the monolayer was calculated with Ohm's
law To adjust for resistance not generated by the monolayer, instrument
calibration was performed with a Vitrogen-coated membrane
¢13
25
2 0 -
1 5 -
1 0 -
5 -
0
0
O
\ ' k ,, ; - - "
,,,' -°
1000
750
500 =
250 ~
RESULTS Mouse gallbladder epithelial cells were dissociated in small sheets
from the gallbladder by treatment with trypsin/EDTA The cells at-
tached to Vitrogen-coated inserts in primary culture when plated over
a confluent layer of human gallbladder myofibroblasts Cells became
confluent in approximately 6 d, and could be successfully passage&
In parallel experiments, mouse gallbladder epithelial cells grown on
Vitrogen-coated plastic plates and fed with conditioned medium
showed attachment and growth, albeit at a slower rate A subcon-
fluent monolayer of cells from cfir ( + / + ) mouse gallbladder is shown
in Fig 1 When the feeder layer was replaced with mouse-derived
flbroblasts [PA317 cells (27)], the cell density was decreased dra-
matically and cells did not become confluent
Cells grown on inserts above a myofibroblast feeder layer formed
confluent monolayers of uniform, short columnar cells consistent with
epithelial cell morphology in cuhure (Fig 2) The cells had distinct
apical and basolateral surfaces with numerous well-developed mi-
crovilli on the apical surface The cells had lobed nuclei, numerous
mitochondria, rough endoplasmic reticula, and well-developed Golgi
bodies In addition, multiple apically localized secretory vesicles
were noted These vesicles were morphologically identical to mucin
granules which stained with periodic acid-Schiff/Alcian Blue, pH
2.5, in a previously characterized canine gallbladder epithelial cell
line (30) No difference in morphology was noted between cfir
( + / + ) (Fig 2 A) and cfir ( - / - ) (Fig 2 B) mouse gallbladder ep-
ithelial cells
The confluency of the monolayer was verified by the ability of the
epithelial layer to act as a barrier to diffusion of the medium from
the upper to lower compartment Co~ffluent monolayers also gener-
ated an electrical barrier between their apical and basolateral sur-
faces Indeed, when mounted in Ussing chambers, monolayers of
wild-type gallbladder epithelial cells exhibited a transepithelial re-
sistance of 163 + 11 DJcm 2 (mean + 1 SE, N = 12)
These cells have been maintained in culture for more than 6
months and 20 passages without discernible changes in microscopic
morphology The cells could be stored in liquid nitrogen and thawed
without changes in morphology
When grown in organotypic culture, the cells maintained strict
polarity with a higher density of well-developed microvilli on the
apical surface The cells grew in monolayers of tall columnar cells
with basally located nuclei as seen under light microscopy (Fig 3)
Electron microscopy (Fig 4) showed cells with numerous lateral in-
Day
FIG 5 Contact inhibition of monolayers with eonfluency The percentage
of cells in S phase decreased as the cell numbers plateaued Closed symbols
represent cfir ( + / + ) cells; open symbols represent cgqr ( - / - ) cells Boxes
and solid lines correspond to percent cells in S phase; circles and dashed lines
correspond to the number of cells/well (× 1000) Each point is a mean +
1 SE of duplicate confluent inserts
tercellular junctions (both tight unctions and desmosomes) and prominent interdigitations between the cells No consistent morpho- logic difference was noted between cfir ( + / + ) (Fig 4 A) and cfir
( - / - ) (Fig 4 B) mouse gallbladder epithelial cells
Preliminary cytogenetic analysis showed that cells retained a kar- yotype of 40 diploid pairs of chromosomes in each cell, the normal chromosome complement of mouse somatic cells (not shown) The flow cytometric profiles of cfir ( + / + ) and cfir ( - / - ) cells were examined with trout erythrocytes as an internal standard Results of flow cytometry of the cells at different days after plating (Fig 5) showed that the percentage of ceils in S phase gradually decreased
to zero as time in culture increased, indicating contact inhibition with confluency
Western blotting with a polyclonal antibody made against the C- terminal sequence of human CFTR confirmed the presence of a broad band at n r 140-180 kDa in cfir (+ / + ) mouse gallbladder epithelial cells (Fig 6) This band was absent in cells from cfir ( - / - ) mice, thus confirming the phenotype of these cells
DISCUSSION Gallbladder epithelium consists of a relatively homogenous pop- ulation of columnar epithelial cells which secrete mucin both con- stitutively and in response to certain secretogogues (21) Culture of canine (30), human (2,17,18,19), and bovine gallbladder epithelial cells (33) has been described Except for the canine cultures, these primary cell cultures do not allow successive passaging leading to long-term culture Other investigators have cultured neoplastic gall- bladder cells (34); the shortcomings of neoplastic or transformed cells are well known One group has reported primary culture of murine gallbladder epithelium but did not report serial passaging (37) We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice
Trang 5108 KUVER ET AL
Q
- - 2 0 0
- - 1 1 6
- - 9 7
- - 6 6
FIG 6 Western blot of gallbladder epithelial cells from cfir ( + / + ) and
cfir ( - / - ) mice with a polyclonal rabbit CFTR antibody Ceils were at Pas-
sage 10 Lane 1: cfir ( + / + ) mouse gallbladder epithelial cells Lane 2: cfir
( - - ] - - ) nlouse gallbladder epithelial cells Lane 3: Molecular weight makers
A prominent diffuse band at Mr 140-180 kDa was present in wild-type cells
and absent in cells from cfir ( - / - ) mice
(20) and compares favorably with the resistance of 57 D-,/cm 2 reported for the prairie dog gallbladder (28) Compared with other cultured cells derived from the digestive tract, this resistance is smaller than the 1,900 D,/cm z generated by colonic T84 cells (29) but is similar
to the 173 ~,Jcm z generated by colonic Caco-2 cells (16) Of partic- ular relevance to the secretory dysfunction(s) of cystic fibrosis, this transepithelial resistance will allow confluent monolayers of gall- bladder ceils to be used in Ussing chambers for studies of ion trans- port and permeability
We have initiated studies with iodide efflux to assess chloride secretion by these ceils (T D Nguyen and R Kuver, unpublished) Mucin synthesis and secretion is also being investigated (23) Trans- duction of these ceils with retroviral vectors is being pursued (R Kuver, S P Lee, W R A Osborne, unpublished), as the gallbladder has been advocated as a target organ for gene therapy (25,43)
In this report we describe a method for the isolation and long-term culture of primary murine gallbladder epithelial cells from wild-type
and cfir ( - / - ) UNC mice The availability of this culture technique
will enable studies of the role of CFTR in cellular function and the mechanisms of mucin synthesis and secretion In addition, with the increasing availability of various types of transgenic and knock-out mice, this culture method may have wider applicability than pro- posed here
These nontransformed cultures, which have been serially passaged
without change in microscopic morphology, provide a model system
for studying the cellular pathophysiology of cystic fibrosis These
cells maintain morphologic criteria of epithelial cells for multiple
passages The presence or absence of CFTR was confirmed with a
polyclonal CFTR antibody on Western blot analysis Thus, these
cells maintained phenotypic differentiation with respect to CFTR
expression Immunocytochemistry for standard epithelial cell mark-
ers was not done due to the lack of availability of antibodies directed
against murine epitopes In addition, the widely available antibodies
are mouse monoclonal antibodies, which in murine tissues lead to
high background staining The morphologic criteria as shown on
monolayer and organotypic culture with both light and electron mi-
croscopy, however, supports a well-differentiated epithelial cell phe-
notype for these cells Although the phenotypic expression of CFTR
on Western blot was noted in cfir ( + / + ) cells at Passage 10, it
remains to be tested whether these cells will retain CFTR and other
epithelial cell phenotypic characteristics with further passaging
The presence of pathologic changes in the gallbladder in CF pa-
tients (1), as well as in the UNC CF mouse model (39), indicates a
correlation between human and murine CF pathophysiology for this
organ Inspissated secretions in the hepatobiliary ductal system of
patients with CF is well described, paralleling changes seen in the
airways, pancreatic ducts, and intestines The relatively homogenous
environment of the epithelium of the gallbladder, with a predominant
columnar epithelial cell type which secretes mucin, provides an ideal
model system for researchers to investigate the relationship between
the complex intracellular functions of CFTR and the pleiotropic clin-
ical manifestations of CF
The formation of a confluent monolayer makes electrophysiological
studies possible Consistent with the presence of tight junctions and
desmosomes seen by electron microscopy, confluent monolayers of
gallbladder cells produced an electrical barrier The transepithelial
resistance of these monolayers, 163 D,/cm z, is similar to the resis-
tance of 136 D-Jcm 2 reported for the Necturus gallbladder epithelium
ACKNOWLEDGMENTS
We thank Audrey Wass for the electron microscopy and Peter Rabinovitch,
MD, PhD, for assistance with flow cytometry This work was supported by a Research Development Program and a Research Fellowship from the Cystic Fibrosis Foundation and NIH grants RO1 DK50246 and P30 DK47754
REFERENCES
1 Anagnostopoulos, D.: Tsagari, N.; Noussia-Arvantitaki, S., et al Gall- bladder disease in patients with cystic fibrosis Eur J Pediatr Surg 3:348-351; 1993
2 Auth, M K H.; Keitzer, R A.; Scholz, M., et al Establishment and immunological characterization of cultured human gallbladder epi- thelial cells Hepatology 18:546-555; 1993
3 Barasch J.; Kiss, B.; Prince, A., et al Defective acidification of intra- cellular organelles in cystic fibrosis Nature 352:70-73~ 1991
4 Bradbury, N A.; Jilling, T.; Berta, G., et al Regulation of plasma mem- brane recycling by C~I'R Science 256:530-532; 1992
5 Cheng, P W.: Boat, T F.: Cranfill, K., et al Increased sulfation of gly- coconjugates by cultured nasal epithelial cells from patients with cys- tic fibrosis J Clin Invest 84:68-72; 1989
6 Clarke, L L.; Grubb, B R." Yankaskas, J R., et al Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr ( - / - ) mice Proc Natl Acad Sci USA 91:479-483; 1994
7 Colledge, W H.; Ratcliff, R.; Foster, D., et al Cystic fibrosis mouse with intestinal obstruction Lancet 340:680: 1992
8 Cuthbert, A W.; Hickman, M E.; MacVinish, L J., et al Chloride se- cretion in response to guanylin in colonic epithelia from normal and transgenic cystic fibrosis mice Br J Pharmacol 112:31-36; 1994
9 Cuthbert, A W.; MacVinish, L J.; Hickman, M E., et al Ion-trans- porting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis Eur J Physiol 428:508-515; 1994
10 De Lisle, R C Increased expression of sulfated gp300 and acinar tissue pathology in pancreas of CFTR ( - / - ) mice Am J Physiol 268 (Gastrointest Liver Physiol 31):G717-G723; 1995
11 Dorin, J R.; Dickinson, P.; Alton, E W F W., et al Cystic fibrosis in the mouse by targeted insertional mutagenesis Nature 359:211-215;
1992
Trang 612 Gray, M A.; Winpenny, J P.; Porteous, D J., et al CFTR and calcium-
activated chloride currents in pancreatic duct ceils of a transgenic
CF mouse Am J Physiol 266 (Cell Physiol 35):C213-C221; 1994
13 Grubb, B R Ion transport across the jejunum in normal and cystic
fibrosis mice Am J Physiol 268 (Gastroiutest Liver Physiol 31):
G505-G513; 1995
14 Grubb, B R.; Vick, R N.; Boucher, R C Hyperabsorption of Na + and
raised Ca2+-mediated C1- secretion in nasal epithelia of CF mice
Am J Physiol 266 (Cell Physiol 35):C1478 C1483; 1994
15 Grubb, B R.; Paradiso, A M.; Boucher, R C Anomalies in ion transport
in CF mouse tracheal epithelium Am J Physiol 267 (Cell Physiol
36):C293-C300; 1994
16 Hidalgo, I J.; Raub, T J.; Borchardt, R T Characterization of the human
colon carcinoma cell line (Caco-2) as a model system for intestinal
epithelial permeability Gastroenterology 96:736-749; 1989
17 Hoerl, B J.; Vroman, B T.; Kasperbauer, J L., et at Biological char-
acteristics of primary cultures of human gallbladder epithelial ceils
Lab Invest 66:243-250; 1992
18 Housset, C.; Carayon, A.: Housset, B., et al Endothelin-1 secretion by
human gallbladder epithelial cells in primary culture Lab Invest
69:750-755: 1993
19 Kobayashi, K.: Kan, M.; Yamane, I., et al Primary culture of human
gallbladder epithelial cells Gastroenterol Japonica 26:363-369;
1991
20 Kottra, G Calcium is not involved in the cAMP-mediated stimulation of
C1- conductance in the apical membrane of Necturus gallbladder
epithelium Eur J Physiol 429:647-658; 1995
21 Kuver, R.; Savard, C E.: Oda D., et al Prostaglandin E generates in-
tracellular cAMP and accelerates mucin secretion by cultured dog
gallbladder epithelial cells Am J Physiol 267:G998-G1003; 1994
22 Kuver, R.; Ramesh, N.; Lau, S., et at Constitutive mucin secretion linked
to CFTR expression Biochem Biophys Res Comm 203:1457-1462;
1994
23 Kuver, R.; Klinkspoor, J H.; Ramesh, N., et al Relationship between
mucin secretion and CFTR in cultured dog and mouse gallbladder
epithelial cells Pediatr Pulm Suppl 12:240; 1995 [Abstract]
24 Leung, A.-Y H.; Wong, P Y D.; Gabriel, S E., et al cAMP- but not
Ca2+-regulated C1- conductance in the oviduct is defective in mouse
model of cystic fibrosis Am J Physiol 268 (Cell Physiol 37):C708-
C712; 1995
25 Maeda, H.; Danel, C.; Crystal, R G Adenovirus-mediated transfer of
human lipase complementary DNA to the gallbladder Gastroenter-
ology 106:1638-1644; 1994
26 Merrick, D.; Blanton, R.; Gown, A., et at Altered expression of prolif-
eration and differentiation markers in human papillomavirus 16 and
18 immortalize epithelial cells grown in organotypic culture Am J
Pathol 140:167-177; 1992
27 Miller, A D.; Rosman, G J Improved retroviral vectors for gene transfer
and expression BioTechniques 7:980-990; 1989
28 Moser, A J.~ Abedin, M Z.; Giurgiu, D I N., et al Octreotide promotes gallbladder absorption in prairie dogs: a potential cause of gallstones Gastroenterology 108:1547-1555; 1995
29 Nguyen, T D.; Canada, A T Modulation of human colonic T84 cell secretion by hydrogen peroxide Biochem Pharmaeol 47:403-407;
1994
30 Oda, D.: Lee, S P.; Hayashi, A Long term culture and partial charac- terization of dog gallbladder epithelial ceils Lab Invest 64:682-692;
1991
31 O'Neal, W K.; Hasty, P.; McCray, P B., Jr., et al A severe phenotype
in mice with a duplication of exon 3 in the cystic fibrosis locus Hum Mol Genet 2:1561-1569; 1993
32 Pilewski, J M.; Frizzell, R A How do cystic fibrosis transmembrane conductance regulator mutations produce lung disease? Curr Opin Puhn Med 1:435 443; 1995
33 Plevris, J N.; Walker, S W.; Harrison, D J., et al Primary culture of bovine gallbladder epithelial ceils Gut 34:1612-1615; 1993
34 Purdum, P P.; Ulissi, A.; ttylemon, P B., et al Cultured human gall- bladder epithelia: methods and partial characterization of a carci- noma-derived model Lab Invest 68(3):345-353; 1993
35 Rabinovitch, P.; O'Brieu K.; Simpson, M., et al Flow cytogenetics Cy- togent Cell Genet 29:65: 1981
36 Rich, D P.: Anderson, M P.: Gregory, R J., et al Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial ceils Nature 347:358-363: 1990
37 Saito, K.; Yoshida, K.; Kohno, N., et al Purification and culture of mouse gallbladder epithelial cells in secondary culture using microexplant culture on collagen gel Nippon Shokakibyo Gakkai Zasshi 89(11):2693-2699; 1992 [Not Translated]
38 Schweibert, E M.; Egan, M E.; Hwang, T.-H., et at CFTR regulates outwardly rectifying chloride channels through an autocrine mecha- nism involving ATP Cell 81:1063-1073: 1995
39 Snouwaert, J N.; Bridgman, K K.; Latour, A M., et al An animal model for cystic fibrosis made by gene targeting Science 257:1083-1088;
1992
40 Snouwaert, J N.; Bridgman K K.; Latour, A M., et at A murine model
of cystic fibrosis Am J Respir Crit Care Med 151:$59-$64; 1995
41 Stutts, M J.; Canessa, C M.: Olsen, J C., et al CFTR as a cAMP- dependent regulator of sodium channels Science 269:847 850;
1995
42 Valverde, M A.; O'Brien, J A.; Sepulveda, F V., et al Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-medi - ated secretogogue-induced volume decrease in small-intestinal crypts Eur J Physiol 425:434-438; 1993
43 Yang, Y.; Raper, S E.; Cohn, J A., et al An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer Proc Natl Acad Sci USA 90(10):4601-4605; 1993