All experiments were approved by the local Institutional Animal Care and Research Advisory Committee as well as by the local government. Cftr
TgH(neoim)Hguwere bred under specified pathogen-free conditions in the isolator unit of the Central Laboratory Animal Facility of the Hannover Medical School. Mice were kept in a flexible film isolator. The temperature within the insulator was maintained at 20–24°C with 40–50% relative humidity. Animals were fed an irradiated (50 kGy) standard chow (SSniff) and autoclaved water (134°C for 50 min) ad libitum. CFTR mRNA analyses were performed on Ztm:MF1, CF/1-Cftr
TgH(neoim)Hgu(generations F8-F10, F18, F32) and CF/3-Cftr
TgH(neoim)Hgu(generations F10, F18, F32) mice at the age of 3 and 6 months. Bioelectrics, Cftr immunoblot and immunocytochemistry were performed on female Ztm:MF1, CF/1-Cftr
TgHeoim)Hgu(generations F28, F30) and CF/3-Cftr
TgH(neoim)Hgu(generations F27, F29) mice at the age of 3 months.
TgH(neoim)Hgumice are available for the scientific community upon written request to HJH.
Generation of inbred Cftr
For the gene targeting and germline transmission to generate the Cftr
TgH(neoim)Hgumice, the embryonal stem cell line E14 was used . E14 originates from the inbred mouse strain 129 (subline 129/Ola-Hsd, abbreviated designation: 129P2) . These cells were injected into blastocysts of the inbred strain C57BL/6J. The resulting chimeric offspring was crossed to C57BL/6J and to the outbred strain MF1 for several times to increase reproduction . By generation F4 the same Cftr
TgH(neoim)Hgumale was mated with two Cftr
TgH(neoim)Hgufemale littermates. Two separate litters were obtained and two animals of each litter became the starting population for the establishment of the two individual inbred Cftr
TgH(neoim)Hguwhich were generated by brother-sister mating for now more than 35 generations. Only one breeding pair per generation was utilized for the continuation of the line. In other words, littermates always shared the same ancestors. Cftr genotyping from tail or ear DNA extractions was conducted by Southern blot hybridization of XhoI/SalI-restricted genomic DNA with the cftr intron 10 probe 1.2 H .
Genotyping of microsatellites
High molecular weight DNA was isolated from spleen tissue (from ten F26 CF/1-Cftr
TgH(neoim)Hgu, ten F26 CF/3-Cftr
TgH(neoim)Hgu, two BALB/c, two C57BL/6 and two DBA/2) . The primers flanking the 105 chosen microsatellite regions were obtained from the MGI database . Microsatellite markers were genotyped in 96 well plates purchased from Greiner, Frickenhausen, pre-coated with 50 ng DNA per well in a Hybaid Thermocycler (Hybaid, Teddington) with a heated lid. One of the two primers per microsatellite was 5'-terminal biotinylated (Invitrogen Ltd). PCR was performed for all microsatellites in a total volume of 30 μl, without oil overlay, using InViTaq polymerase (InViTek, Berlin) according to the instructions by the manufacturer. Following PCR an 8 μl aliquot was transferred to a multiwell plate and allowed to dry overnight at 37°C, dissolved in 10 μl loading buffer (0.2% w/v xylenecyanol and bromphenolblue in formamide) and denatured for 5 min at 95°C. The PCR products were separated by direct blotting electrophoresis (GATC 1500, MWG Biotech, Ebersberg, Germany) on a denaturing acrylamide gel (4% acrylamide/N,N'-methylenebisacrylamide 29:1 containing 6 M urea in 0.9 M Tris-0.9 M boric acid-0.02 M EDTA buffer) and simultaneously transferred to a Hybond N+membrane (Amersham). Signals were visualised by blocking the membrane in 1.5% (w/v) of blocking reagent in Buffer 1 (100 mM Tris-HCl, 150 mM NaCl, pH 7.5), followed by incubation in diluted solution of anti-biotin alkaline phosphatase conjugate in Buffer 1. The membrane was further washed three times with 1% Triton X-100 in Buffer 1 and equilibrated for 15 min in assay buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The membrane was covered for 5 min with reaction buffer containing 10%(v/v) Sapphire II (Tropix) and 600 μl CDPstar (Tropix) (12.5 mM) in 50 ml assay buffer, followed by rinsing with a solution containing 10% v/v Sapphire II and 60 μl CDPstar (12.5 mM) in 50 ml assay buffer. Signals were exposed to Kodak XA-R films. Evaluation of results was performed as described by Mekus et al .
Isolation of RNA and quantitative RT/PCR
All tissue samples from Ztm: MF1 and F8–10 as well as F18 CF mice were immediately frozen in liquid nitrogen after extirpation and stored at -70°C until used. Prior to the isolation of RNA, all solutions and plasticware were treated for at least 12 h with 0.04% (by vol.) aqueous diethyl pyrocarbonate solution. RNA was isolated essentially as described by Chomczynski and Sacchi . Briefly, tissue was ground with a pestle in a mortar under liquid nitrogen and then homogenized in denaturing solution (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 200 mM β-mercaptoethanol). The homogenate was mixed sequentially with 1 M sodium acetate (pH 4), phenol and chloroform/isoamyl alcohol; the mixture was centrifuged and the RNA removed from the upper aqueous phase. Following isopropanol precipitation, the RNA pellet was redissolved in denaturing solution, reprecipitated with isopropanol, washed with ethanol, dried, dissolved in DEPC-treated water and stored at -70°C until used. The quality of the preparation was checked by UV spectrophotometry and ethidium bromide stain of 2 μg RNA separated by 1% agarose gel electrophoresis. The absence of contamination by DNA in the RNA preparation was verified by PCR with oald141N 5'-GGCAAGGGCATCCTGGCTGCAGA and oald581I 5'-TAACGGGCCAGAACATTGGCATT. These oligonucleotide primers yield a 421-bp product for both murine genomic DNA and reverse-transcribed murine RNA (PCR conditions per cycle: annealing at 62°C, synthesis at 72°C, denaturation at 92°C 60 s each).
For cDNA synthesis, 8 μg denatured RNA were added to the 40-μl reaction volume of 40 U avian myoblastoma virus reverse transcriptase, 120 μg/ml oligo(dT)15, 2000 U/ml RNasin, 0.9 mM dNTPs, 10 mM DTT, 75 mM KCl, 3 mM MgCl2, 50 mM Tris/HCl, pH 8.3, incubated for 1 h at 37°C, distributed in 10 μl aliquots and stored at -70°C for a maximum of one month. The differential amounts of total cftr mRNA and correctly spliced wild-type cftr mRNA transcripts in specimens from CF/1-Cftr
TgH(neoim)Hgumice were determined by quantitative PCR kinetics modifying a protocol described by Hoof et al . Two oligonucleotide primer pairs A1–A2, B1–B2 were designed upstream of and encompassing the neo insertion site  (A1 mmcftr 1164–1189 5'-GCATTGTCCTACGTATGTCAGTCACG; A2 mmcftr 1524–1499 5'-CCTAGTCCAGTAGATCCAGTAATAGC; B1 mmcftr 1499–1524 5'-GCTATTACTGGATCTACTGGACTAGG; B2 mmcftr 2053–2032 5'-GCTCGGACGTAGACTTTGTAGC) (GenBank Accession Number: NM_021050).
Amplification of Cftr cDNA stretches by PCR with these primer pairs showed an identical kinetics of product formation (Δcmax/cycle: ± 0.4% intestine; ± 1.3% lungs) with Ztm:MF1 reverse-transcribed control RNA (PCR conditions per cycle for both primer pairs: annealing at 58°C, synthesis at 72°C, denaturation at 92°C 60 s each). For quantitation of mRNA, the kinetics of cDNA formation was monitored by incorporation of 1 × 105 Bq [α-33P]dATP (> 1 × 1014 Bq/mmol) into cDNA. The PCR assay (50-μl reaction volume covered with 60 μl mineral oil) contained 0.5 μM of each oligonucleotide primer, 0.2 mM dNTP, 3 μl of oligoT-reverse transcribed reaction mix (see above), 1.3 μl DMSO, 1.5 U Taq DNA polymerase (InviTAQ), 7.5 mM MgCl2, 16 mM (NH4)2SO4, 50 mM Tris/HCl, pH 8.8, 0.1% Tween.
Aliquots of the same oligoT-reverse transcribed reaction mix were amplified in parallel in separate tubes in the same blocks of the thermocycler with primer pairs A1–A2, B1–B2 and oald141N-oald583I, respectively. During the exponential phase of the amplification of the respective cDNA aliquots of 10 μl were withdrawn in 2-cycle intervals and separated by 5% PAGE. The dried gel was exposed to a Fuji imaging plate type BAS-IIIs in order to quantify the yield of cDNA product by photoimaging signal. For each RNA preparation, the optimal window of PCR kinetics had been determined for each of the three cDNA sequences in a prior pilot PCR. For quantification of the cftr mRNA contents in the samples, data sets for each murine tissue were normalized to its invariant aldolase mRNA contents in order to adjust to the variable quality of mRNA template in the RNA preparations and assay-to-assay variability of the reverse transcription. This approach was verified by the observation that the repeated preparation of RNA from one tissue specimen showed similar variations in aldolase cDNA kinetics as tissue RNAs from unrelated animals. The quantitative amount of cftr mRNA in the various samples was calculated from the complete data set for one tissue whereby the experimental curves log [cDNA] vs. PCR reaction cycle n were converted into calibration curves cftr RNA molecules vs. PCR cycle for various yields of cDNA . Only those datasets were evaluated that within experimental error had given indistinguishable Cftr cDNA formation kinetics with primer pairs A1–A2 and B1–B2 for reverse-transcribed Ztm:MF1 RNA (positive control). Cftr mRNA contents of each RNA preparation was assayed at least in triplicate.
In case of the analysis of F32 CF/1-Cftr
TgH(neoim)Hguand F32 CF/3-Cftr
TgH(neoim)Hguanimals the tissues (duodenum, jejunum, ileum, nose) were stored in RNAlater (Ambion) after extirpation and stored at -20°C until use. RNA was extracted using the RNAeasy mini kit (Qiagen) following the instructions of the manufacturer. Extracted RNA was stored at -80°C until use. The amount of aldolase and correctly spliced Cftr mRNA was determined by semiquantitative RT/PCR kinetics. We titrated for the first reaction cycle, when the gel-separated cDNA became visible by ethidium bromide fluorescence during the late exponential phase of PCR . This procedure yielded an estimate of mRNA concentration within half an order of magnitude, whereas the interexperimental error of the quantitative PCR on mRNA contents in F8–10 and F18 CF mice that measured the incorporation of labelled nucleotide during the exponential phase of PCR was twofold or less.
Following RT-PCR cDNA products were sequenced by Qiagen GmbH.
Western blot analysis
Wild type MF1 mice and CF/1-Cftr
TgH(neoim)Hgumutant mice were anaesthetized with a hypnorm/diazepam mixture. Their abdomens were opened and their small intestines dissected. Epithelial cells originating principally from the villus region were isolated at 0–4°C from the jejunum by everting the intestinal segments on metal rods attached to a vibration apparatus (Vibromixer type E1 from Chemap A.G.) and exposing them to vibration (50 Hz, amplitude 1.5 mm) for 30 min in 0.14 M NaCl containing 5 mM EDTA pH 7.4. Detached jejunal enterocytes were collected by centrifugation at 800 g for 15 s and suspended in 10 ml of a medium containing 12 mM Tris-HCl pH 7.4, 0.3 M mannitol, 10 mM KCl, 0.5 mM EDTA and a protease-inhibitor cocktail containing 0.3 mM Pefablock (La Roche), 10 μg/ml aprotinine, 5 μg/ml leupeptine, 1 μg/ml pepstatin A, 1 μg/ml chymostatin, 50 μg/ml soybean trypsin inhibitor and 0.03 g/l phosphoramidon. Vesiculation of intestinal membranes was achieved by a freeze-thaw procedure described initially for rat enterocytes and crude microsomal membranes were isolated from half of the cell lysate by a two-step differential centrifugation procedure (10 min, 4000 g, followed by 60 min, 40 000 g). The other half was used to isolate BBMV by differential precipitation with 10 mM MgCl2 and differential centrifugation (15 min, 3000 g followed by 30 min, 27 000 g) essentially as described by . The membrane pellets were solubilized by vortexing in 30 μl modified Laemmli sample buffer [0.06 M Tris-HCl; 2% (w/v) SDS, 10% (w/v) glycerol, 0.1 M dithiothreitol, 0.1% (w/v) bromophenol blue and the protease inhibitor cocktail, pH 6.8] and incubated for 30 min at room temperature. Following centrifugation (2 min, 8000 g) samples of the supernatant (10 μl, adjusted to 20 μg protein) were separated on 6% polyacrylamide slab gels using a Bio-Rad Miniprotean apparatus (Bio-Rad Laboratories). Proteins were subsequently electroblotted onto nitrocellulose paper (0.1 μm pore size; Schleicher and Schuell) in 0.025 M Tris, 0.192 M glycine, 20% (v/v) methanol. The blots were incubated overnight at 4°C with 0.02 M Tris-HCl, 0.15 M NaCl, 0.1% (w/v) Tween 20 pH 7.5 (TTBS), followed by overnight incubation at 4°C with a 1:1000 dilution of affinity-purified anti-CFTR antibody R3195 in TTBS. Blots were washed three times in TTBS, incubated with peroxidase-conjugated anti-rabbit IgG (Tago Inc.; 1:3000 in TTBS for 2 h), and washed four times with TTBS. Peroxidase activity was detected with bioluminescence reagent (ECL kit; Amersham) on X-ray film, and CFTR bands were quantitated with the Molecular Imaging System GS-363 (Bio-Rad).
The rabbit polyclonal antibody R3195 was raised against a thyroglobulin-conjugated 13 amino acid COOH-terminal peptide sequence of rodent CFTR and was affinity purified on a peptide epoxide-activated Sepharose column, eluted with 4.9 M MgCl2, dialysed and concentrated. CFTR labelling specificity has been demonstrated previously in western blot and immunocytochemical assays by the loss of immunostaining in tissue specimens from CFTR-/- mice .
Intestine: Wild type (MF1 and C57BL/6J) and mutant CF/1-Cftr
TgH(neoim)Hgumice were sacrificed by cervical dislocation, the intestine was dissected and the jejunum, duodenum and ileum were rinsed with ice-cold saline and fixed in 3% (w/v) paraformaldehyde for 16 h, prior to standard paraffin embedding. Sections (5 μm) were deparaffinised, followed by microwave treatment in 0.01 M sodium citrate solution according to Devys et al. . Endogenous peroxidase activity was blocked by a 30 min pre-incubation in 0.1 M PBS, 0.6% (v/v) H2O2 and 0.12% (w/v) sodium azide. Subsequently, sections were incubated with antibody R3195 (1:100) at room temperature for 1.5 h followed by a 60 min incubation with a peroxidase-conjugated secondary antibody. Enzymatic detection of antigen-antibody complexes was achieved by incubation in substrate solution containing H2O2 and 3,3'-diaminobenzidine tetrahydrochloride (DAB; Serva). Finally, the sections were counterstained with haematoxylin. Labelling specificity was verified by incubations without primary antibody. In both cases, background labelling appeared negligible.
Nasal: Freshly excised nasal tissue from wild type MF1 and C57BL/6J and mutant CF/1-Cftr
TgH(neoim)Hgumice was used for immunocytochemical analysis. The tissue was treated as described for the intestine.
Deparaffinised sections (5 μm) were stained with Alcian blue (1% w/v in 3% v/v acetic acid, Sigma) for 30 min at RT, rinsed in tab water and counterstained with Nuclear fast red (Sigma) for 10 min.
Short-circuit current measurements
Freshly excised mouse ileum and nasal epithelium of CF/1-Cftr
TgH(neoim)Hguhomozygous animals were used for short-circuit current (Isc) measurements and compared with wild type controls. All experiments were done in duplicate, n = the number of mice used. Experiments were performed at 37°C. The basic perfusion solution (modified Meyler's solution) consisted of 105 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 20.2 mM NaHCO3, 0.4 mM NaH2PO4, 10 mM HEPES, saturated with 95% O2 and 5% CO2, pH 7.4.
Mouse ileum was excised under hypnorm/diazepam anaesthesia and reverted on a plastic rod. The muscle layer was cut longitudinally using a blunt razor blade and was stripped of fat manually. The stripped tissue was mounted in a holder with the mucosal side up (exposed tissue area 0.2 cm2). After insertion of the holder into the Ussing chamber the tissue was allowed to recover for 10–20 min and to reach a stable baseline. To the serosal side of the ileum glucose (10 mM) and indomethacin (10 mM) were added. After equilibrium, the following compounds were added consecutively to the mucosal (M) or serosal (S) side of the tissue: Forskolin (10 μM, S), genistein (100 μM, M+S) and carbachol (200 μM, S). All compounds were present throughout the experiment.
Mouse nasal epithelium was isolated as described by Grubb et. al. . In brief, the mice were sacrificed by cervical dislocation and the skin of the head was peeled back in order to reach the underlying paired nasal bones. These were removed and the two sheets of the nasal epithelia, separated by the septum, were isolated independently. The sheets of epithelia were mounted immediately between the Ussing chambers (exposed area 1.13 mm2). The chambers were filled with gassed modified Meyler's solution supplemented with glucose (10 mM) and indomethacin (10 mM) and the short circuit measurements were started. After equilibration amiloride (10 μM, M) was added followed by forskolin (10 μM, after stabilization of the current).
P-values were determined by using the student's t-test. P-values < 0.05 were considered significant.