1 Department of Pharmacology, Gallbladders
from cystic fibrosis (CF) mice
(Cftrtm1Cam and
Cftrtm2Cam)
were examined with the short-circuit current technique.
The tissues failed to show any electrogenic anion transport in response
to forskolin (cAMP stimulus) but responded to the
Ca2+ ionophore ionomycin.
Administration of the plasmid pTrial10-CFTR2 complexed with cationic
liposomes
{3
cystic fibrosis transmembrane conductance regulator; luciferase; THE MOUSE GALLBLADDER secretes
HCO Animals
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-[N-(dimethylaminoethane)-carbamoyl]cholesterol and
L-
-phosphatidylethanolamine
dioleolyl} to the airways restored the phenotype of CF
gallbladders to that of the wild type, but did not do so when given
orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the
reporter genes pCMV-luc and
pCMV-LacZ, we showed that 1) the intratracheal route was more
effective than the oral, intravenous, intramuscular, subcutaneous, or
intraperitoneal routes in expressing luciferase activity in the
gallbladder and 2)
-galactosidase staining after pCMV-LacZ was confined
to the columnar epithelium lining the gallbladder without any
discernible activity in its smooth muscle. The discovery of an unusual
route for gene transfer to the biliary system may give useful insight
into counteracting the consequences of biliary fibrosis in human CF
patients.
-galactosidase
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
3 predominantly through a
conductive anion pathway in response to a cAMP stimulus (14). To
determine if the conductive anion channel was the cystic fibrosis
transmembrane conductance regulator (CFTR), we used transgenic animals
in which functional CFTR is not present to examine how this affects
secretion. We used both cystic fibrosis (CF) null mice
(Cftrtm1Cam),
in which no CFTR is produced, and
F508 mice
(Cftrtm2Cam),
which produce a mutant protein that is not incorporated in the plasma
membrane (3, 16). Using forskolin, we found that murine CF gallbladders
show virtually no response to a cAMP stimulus. In humans,
both the mRNA for CFTR and the protein
itself are found in the intra- and extrahepatic biliary epithelium,
including the gallbladder, where the protein is located at the apical
face of the cells (2, 8). In CF there are primary abnormalities in
hepatobiliary anion transport. Seventy percent of CF adults have focal
biliary fibrosis, which may lead to multilobular biliary cirrhosis and
portal hypertension (4). After a chance observation made using
gallbladders from CF mice, we showed that the murine gallbladder
readily takes up genetic material expressed in the columnar epithelial
cells. When a plasmid-liposome complex (lipoplex) containing the cDNA
for human CFTR is instilled
intratracheally in CF animals, a phenotypic reversion occurs in the
gallbladder and ion-transporting activity is restored. The
demonstration of gene transfer in the CF mouse gallbladder in vivo may
provide useful insights for humans.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
F508 CF mice
(Cftrtm2Cam)
(3), aged between 1 and 3 mo, were used in the study. The care of the
animals, the method of preparing gallbladders for short-circuit current
(Isc)
recording, and the composition of the Krebs-Henseleit solution (KHS)
were as previously described by Martin et al. (14).
Isc Recording
Gallbladders were mounted in Ussing chambers with a window area of 4 mm2 and bathed on both sides with KHS (20 ml) warmed to 37°C, continually circulated by bubbling with 95% O2-5% CO2. The tissues were short circuited with a World Precision Instruments dual-voltage clamp with series resistance compensation, and Isc was recorded continuously with a MacLab and associated AppleMac computer.Preparation of Lipoplexes
pTrial 10-CFTR2.
For tracheal transfection, 10 µg of plasmid (pTrial10-CFTR2)
containing the cDNA for human CFTR
(12) were added to 25 µl of Krebs-HEPES buffer (KHB; pH 9) to which
was added 100 nmol of
3-[N-(dimethylaminoethane)-carbamoyl]cholesterol
(DC-Chol) and
L-
-phosphatidylethanolamine
dioleolyl (DOPE) (3:2 molar ratio) liposomes mixed with 15 µl of KHB
(pH 9). The plasmid and liposomes were mixed to give a
total volume of 100 µl, and complexes were allowed to form at
37°C for 10 min in a polystyrene tube. Control plasmid
pTrial 10 was prepared in the same way for transfection.
pCMV-luc and pGL-3-luc. For tracheal or oral transfection 10 µg of pCMV-luc plasmid mixed with 25 µl KHB (pH 9) were added to 100 nmol of DC-Chol and DOPE liposomes in 15 µl KHB. As described above, the complex was allowed to form at 37°C for 10 min in a total volume of 100 µl. Likewise, for other routes 133 µg of pCMV-luc plasmid were added to 560 nmol of DC-Chol and DOPE in KHB and the complex was allowed to form. pGL-3-luc was used in the initial experiment in which nasal and oral routes were compared. pGL-3-luc (5 µg DNA) was mixed with liposomes in the same proportions as for pCMV-luc.
pCMV-LacZ. We mixed 133 µg of plasmid (pCMV-LacZ) with 560 nmol DC-Chol and DOPE liposomes with 5% glucose and allowed the complex to form at 37°C for 10 min in a polystyrene tube.
Administration of Lipoplexes to Mice
Mice were anesthetized with bromethol (0.02 ml/g body wt), and the transfection mixture was instilled into the trachea, 20 µl at a time (for pTrial10-CFTR2 and pCMV-luc, 10 µg DNA for each) until the whole volume (100 µl) had been delivered. For nasal transfection, one-half the tracheal dose (pTrial10-CFTR2, 5 µg) was given, divided between the two nostrils. Anesthetized mice were placed on their backs, and 2 µl transfection mixture were placed in turn over each nostril until all the mixture was given. For oral transfection, 5 or 10 µg DNA (as pTrial10-CFTR or pCMV-luc lipoplexes) were given directly into the stomach in anesthetized mice. We gave 133 µg DNA (as pCMV-luc lipoplex) by other routes in anesthetized mice as follows: intramuscularly into the hindlimb, into the peritoneal cavity, subcutaneously under the skin of the back, and intravenously. In the latter case, the lipoplex was administered over a 15-min period via the tail vein, using a pediatric cannula. pCMV-LacZ lipoplex was also given by the intravenous route as for pCMV-luc. With all routes, the mice were allowed to recover for 2 days before use.Demonstration of Gene Transfer to the Gallbladder
Along with demonstrating function in transfected gallbladders, we investigated the formation of mRNA for CFTR. Furthermore, reporter genes were used to explore the location and extent of protein formation when different routes for transfection were used. Experiments with reporter genes (forDetection of mRNA by RT-PCR. Total mRNA was extracted from a single gallbladder (3-5 mg) using the acid guanidinium thiocyanate method (1). cDNA was produced by reverse transcription from ~5 µg of total RNA with Moloney murine leukemia virus RT (GIBCO-BRL) using a pd(N)6 primer. PCR of the cDNA templates was performed using a pair of specific primers for human CFTR that amplified a fragment of 500 bp containing exons 7-10. The primers were X7H, 5'-ACAAACATGGTATGACTCTCTTGG-3', and X10H, 5'-GTTGGCATGCTTTGATGACGCTTC-3'. The pTrial10-CFTR2 plasmid served as a positive control for the human primers. The PCR reactions were carried out in 20 µl of PCR buffer containing dATP, dTTP, dCTP, and dGTP at a concentration of 1 mM, 10 pmol of each primer, 1 U Taq polymerase (Advanced Biotechnologies), and 5 µl of cDNA. The reaction mixtures were heated to 95°C for 5 min in a Techma PHC-3 PCR machine and were then subjected to 40 cycles of denaturation (94°C for 30 s), primer annealing (60°C for 30 s), and extension (72°C for 45 s). PCR products were examined by electrophoresis on a 1% agarose gel stained with ethidium bromide. To ensure that the human CFTR signal from transfected gallbladders subjected to RT-PCR was not a result of amplifying residual plasmid pTrial10-CFTR2 cDNA, the extracted material was subjected to DNase treatment as described below.
Gallbladder total RNA (~5 µg) was treated with 40 U of RNase-free DNase (Promega) in RT buffer in the presence of 10 U RNase inhibitor (Promega) for 1 h at 37°C. Afterward, 0.1 vol of ice-cold 3 M sodium acetate and 2 vol of ice-cold ethanol were added, mixed, and left for 1 h atLuciferase formation after transfection by different routes.
To measure luciferase activity, the gallbladders were snap frozen on
dry ice immediately after removal from the mouse. Each gallbladder was
separately homogenized in 75 µl of reporter lysis buffer (Promega),
using a Polytron homogenizer, and vortexed for 15 s. The homogenates
were centrifuged at 4°C to give a clear supernatant, the latter
being stored at 70°C until assayed. To assay, 20 µl of
each extract were warmed to room temperature and 100 µl of luciferase
assay reagent (Promega) were added and gently mixed. Light emission was
measured in a Turner luminometer (model 20) using a 3-s delay and 30-s
integration time. The protein content of the extracts was measured at
OD595nm using a Bio-Rad protein assay system.
-Galactosidase staining.
Gallbladders were washed in PBS (0.1 M, pH 7.4) and fixed in 0.4%
formaldehyde for 30 min at room temperature. After further washing in
PBS, the gallbladders were stained overnight at 30°C in a solution
of 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
potassium ferri- and ferrocyanide. Gallbladders were then paraffin
embedded, and 6-µm sections were cut and counterstained.
Statistical Treatment of Results
Student's t-test was used to test for the significance of observed differences, with P < 0.05 considered significant. When F values indicated that the standard deviations of the two populations being compared were significantly different, a nonparametric test (Mann Whitney U-test) was used to compare means. ![]() |
RESULTS |
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Ion Transport in Gallbladders from CF Mice
Gallbladders from CF mice, both null and those with the
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In all experiments in which CF mice were used, a single colonic epithelial preparation was used as a check on the genotyping of the animals. In the murine CF colon, a small K+ secretory response to forskolin is revealed, which is reversed by furosemide (6). Reference has already been made to the economy required in the use of CF mice, a strategy that on one occasion led to unexpected serendipity. A gallbladder taken from a CF null animal was found to have a perfectly normal wild-type phenotype, whereas the colonic epithelium from the same animal had a characteristic CF profile (Fig. 3A). On this occasion, the mouse had been transfected with pTrial10-CFTR2 complexed with cationic liposomes by intratracheal instillation 2 days earlier. The transfection was carried out as part of another study (12). In all, four similarly treated animals were examined in the same way, and the pooled results are depicted in Fig. 1B, where a phenotypic fingerprint remarkably similar to wild-type animals is revealed. Three other CF mice were transfected by the nasal route, and the gallbladders of two of the three animals showed a wild-type phenotype, one of which is shown (Fig. 3B). Finally, three CF mice were transfected with pTrial10-CFTR2 lipoplex (10 µg DNA) given by the intragastric route. No indication was found that the phenotype of the gallbladders was other than expected from the genotype in these three, one of which is depicted in Fig. 3C. Finally, two CF null mice receiving pTrial10 (i.e., plasmid without CFTR cDNA) by the tracheal route yielded gallbladders with a CF phenotype.
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Demonstration of Human CFTR mRNA in Transfected Murine Gallbladders
Total RNA from one gallbladder transfected via the trachea 2 days previously was subjected to RT-PCR as described. A band corresponding with the positive human CFTR control was found in the transfected sample (Fig. 4, left). Although it seemed unlikely that plasmid DNA could be present in the gallbladder 2 days after transfection with pTrial10-CFTR2 in the airways, a further experiment was devised to test for this possibility. A fresh gallbladder from a null mouse, transfected 2 days before, was extracted and treated with DNase as described above, before being subjected to PCR. The absence of a signal corresponding to the positive control indicated that either no plasmid DNA was present or that the DNase treatment had been effective. The remaining RNA was then subjected to RT-PCR and gave a product corresponding to the positive control, with no signal when either the RT step was omitted or no cDNA was added. To confirm the integrity of the mRNA, the same sample was subjected to RT-PCR, using primers for the ubiquitous proliferating cell nuclear antigen (PCNA), which gives a 720-bp fragment encompassing exons 1-4 of mouse PCNA.
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Luciferase Expression in Gallbladders Transfected with pCMV-luc or pGL-3-luc
To further investigate the route from the lungs to the gallbladder, we investigated whether the gallbladder could be transfected by the oral route using pGL-3-luc. The same amount of plasmid was given by the nasal and oral routes, the latter being equivalent to the whole of the airway dose being swallowed. There was no significant difference between the background signal from animals receiving liposomes only and the light emitted by gallbladder extracts from orally transfected animals, whereas the intranasal route was effective in stimulating luciferase activity in the gallbladder (Fig. 5A). In a more elaborate experiment, groups of wild-type mice were transfected with pCMV-luc lipoplex by five different routes, namely tracheal, intravenous, intramuscular, intraperitoneal, and subcutaneous, as described in MATERIALS AND METHODS. Control animals were given liposomes without plasmid. Two days later the mice were killed, the gallbladders were removed, and tissue extracts were prepared for assay by light emission. The results from this experiment are shown in Fig. 5B. Light emission from tissue extracts from animals given the plasmid via the intramuscular, intraperitoneal, or subcutaneous routes showed no increase over background values, whereas values from the intravenous recipients were over five times that of the background. However, even these values were small compared with the values that were obtained when the plasmid was delivered intratracheally, which were 100 times greater than the background value.
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Histological Demonstration of Gene Transfer to the Gallbladder
To further explore gene transfer to the gallbladder, the plasmid pCMV-LacZ (10 µg), complexed with cationic liposomes, was given intratracheally, but no staining for
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DISCUSSION |
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Gallbladders from CF mice, either in CF null (16) (producing no CFTR)
or homozygous F508 mice (3) (producing a nonfunctional mutant form
of CFTR), are incapable of generating an electrogenic anion secretory
response to forskolin, in contrast to wild-type gallbladders (Fig. 1).
This finding, together with those by Martin et al. (14), indicates that
CFTR is essential for electrogenic HCO
3 secretion in the murine
gallbladder. Very similar conclusions were made for
HCO
3 secretion in normal and CF human
airway epithelia (17). Here HCO
3 secretion via an apical conductive pathway was demonstrated in response
to cAMP, which was absent in CF epithelia.
Both types of CF gallbladders showed Isc increases that were often transient and followed by an elevated plateau to the Ca2+ ionophore ionomycin, a finding again consistent with the study on CF airway epithelia by Smith and Welsh (17). The normal mouse gallbladder also shows responses to the Ca2+ ionophore ionomycin, but we have not been able to demonstrate statistically clear differences between wild-type and CF tissues. However, similar to the human gallbladder (8), the mouse epithelium is sensitive to both cAMP and Ca2+ signals.
In summary, the anion secretory current in the gallbladder is CFTR
dependent, consistent with earlier studies (15, 18), in which
Cl was identified as the
transported species. In these earlier studies (15, 18), reliance was
placed on the failure to elicit a secretory response in normal
gallbladders when all Cl
was removed from both bathing solutions. It is not extraordinary to
suggest that CFTR can allow ions other than
Cl
to permeate, as there is
ample evidence from patch-clamp studies that CFTR channels are
permeable to HCO
3. For example, in the
pancreatic duct the permeability ratio of
HCO
3 to
Cl
is 0.13 (9), whereas in
the cortical collecting duct the ratio reaches 0.67 (11).
The limited availability of CF mice encouraged us to examine gallbladders from mice that as part of another study had been transfected intratracheally or nasally with pTrial10-CFTR2 complexed with cationic liposomes. Mice are genotyped by extracting the DNA from tail clips before experiments, but we always examine the colonic epithelium to confirm this. The expectation was that the gallbladders of transfected mice would behave as tissues with the CF phenotype, yet a normal phenotype was observed, with responses to forskolin and acetazolamide. Meanwhile, the colonic epithelia were unchanged, presenting the classic CF phenotype (6). No responses to forskolin were seen in gallbladders from mice transfected intragastrically. Although this latter result does not preclude the possibility that the oral route may occasionally be successful, it seems unlikely that the reversal after airway transfection was caused by the mice swallowing plasmid introduced into the airways. While functional studies alone provided a powerful piece of evidence for the pulmonary route for successful transfection of the biliary tract, the demonstration of the presence of human CFTR mRNA in gallbladder tissue added a vital confirmation.
Using wild-type mice and the pCMV-luc
lipoplex, we further explored the transfer of genetic information to
the gallbladder. Again, as with pTrial10-CFTR2, it was necessary for
the plasmid to be both transcribed and translated to give luciferase
expression and hence light emission. Strikingly, only the intratracheal
route gave good transfection of the gallbladder, although there was an
increase in activity after intravenous infusion, while other routes
were ineffective. Although transfection of the gallbladder by the
intratracheal route was significantly better than with the other
routes, there was considerable variability between animals, as
evidenced by the large standard error (Fig. 5). This seems to indicate
that there are many factors determining gene transfer, which are, at
present, unknown and uncontrolled. It was disappointing not to be able
to demonstrate the transfer of the
pCMV-LacZ lipoplex to the gallbladder
via the intratracheal route. It has been estimated from staining and
immunologic studies that reliance on the appearance of a blue stain is
43 times less sensitive than detecting the presence of
-galactosidase using immunostaining (5). Thus the demonstration of
-galactosidase staining is a rather insensitive method, while
phenotypic correction measured by bioassay requires only low level
expression. For instance, CF "knockout" mice expressing only 10%
of the normal CFTR have higher survival rates compared with CF null
mice and show minor functional responses (7). By increasing the dose of
pCMV-LacZ plasmid some 10-fold,
staining was demonstrated after delivery by the intravenous route and
confined to the gallbladder epithelium. No staining of the underlying
smooth muscle was discernible, although it cannot be concluded that it was completely absent.
Others have shown (10) that it is possible to transfect human CF intrahepatic biliary epithelial cell lines with adenoviral vectors and restore cAMP-dependent halide efflux. Furthermore, biliary epithelial cells in the rat can also be transfected by retrograde infusion of adenoviral vectors into the common bile duct (19). The latter approach involves an invasive procedure and a more efficient gene delivery system. Transfection with liposomes, either in vivo or in vitro, is generally considered to be an inefficient method for gene transfer even though, for human gene therapy, it is less likely to result in immunologic consequences on repeated application (13). The serendipitous finding that the gallbladder is relatively easily transfected by material given via the airways is not only an important finding in relation to gene therapy for CF, but when the mechanism is unraveled it may have important lessons for improving the efficiency of transfection in other situations.
In summary, it is found that the cAMP-dependent anion secretory
current, due primarily to the secretion of
HCO3, is absent in murine CF
gallbladders. It is concluded that CFTR is essential for the
HCO
3 secretory current and that gene
transfer via an airway transfection route can restore the secretory
activity in CF gallbladders.
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ACKNOWLEDGEMENTS |
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This work was supported by a Sir Henry Wellcome Commemorative Award for Innovative Research to A. W. Cuthbert. Supplies of mice, plasmids, and other materials were supported by grants from the Medical Research Council and the Cystic Fibrosis Trust.
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FOOTNOTES |
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Address for reprint requests: A. W. Cuthbert, Dept. of Pharmacology, Univ. of Cambridge, Tennis Court Rd., Cambridge CB2 1QJ, United Kingdom.
Received 29 October 1997; accepted in final form 3 February 1998.
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