Human Molecular Genetics, 2002, Vol. 11, No. 3 243-251
© 2002 Oxford University Press
The severe G480C cystic fibrosis mutation, when replicated in the mouse, demonstrates mistrafficking, normal survival and organ-specific bioelectrics
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK, 1Department of Gene Therapy, National Heart and Lung Institute at Imperial College, London, UK, 2CBG-Department of Clinical Genetics and 3Department of Biochemistry, Faculty of Medicine and Health Sciences, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Received September 13, 2001; Revised and Accepted November 23, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The majority of cystic fibrosis patients produce a mutant form of CFTR (
F508) which has been shown to be mislocalized in both humans and mice. G480C, another clinically ‘severe’ mutation, has also been demonstrated to be defective in its intracellular processing, but when allowed to traffic in Xenopus oocytes showed similar channel characteristics to that of wild-type CFTR. We have replicated the G480C mutation in the murine Cftr gene using the ‘hit and run’ double recombination procedure. As expected, the G480C cystic fibrosis mouse model expresses the G480C mutant transcript at a level comparable to that of wild-type Cftr. The homozygous mutant mice were fertile, had normal survival, weight, tooth colour and no evidence of caecal blockage, despite mild goblet cell hypertrophy in the intestine. Analysis of the mutant protein revealed that the majority of G480C CFTR was abnormally processed and no G480C CFTR-specific immunostaining in the apical membranes of intestinal cells was detected. The bioelectric phenotype of these mice revealed organ-specific electrophysiological effects. In contrast to F508 ‘hit and run’ homozygotes, the classic defect of forskolin-induced chloride ion transport is not replicated in the caecum, but the response to low chloride in the nose is clearly defective in the G480C mutant animals. The mild phenotype of these G480C mutant animals combined with the defective chloride transport in the nose uniquely provides a valuable resource to test novel pharmacological agents aimed at improving trafficking and correcting the electrophysiological defect in the respiratory tract. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cystic fibrosis (CF) is a fatal autosomal recessive genetic disease characterized by abnormal epithelial ion transport. The mutated gene responsible is the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a protein which functions among other things as an epithelial cAMP-regulated Cl– ion channel (1). Many hundreds of mutations have been described in CF patients but the most common mutation is caused by deletion of a phenylalanine at position 508 (
F508) (2). This mutation causes misfolding of the protein, incomplete glycosylation and prevents correct trafficking of the protein to the apical plasma membrane (3).
In humans, F508 accounts for 
70% of all mutant CF alleles and is considered ‘severe’ with respect to CF phenotype. Other ‘severe’ mutations have been described and G480C is one where an amino acid substitution occurs in exon 10 of CFTR (3). Both G480C and F508 mutations show a primary defect in protein processing and trafficking, such that mutant protein is retained and degraded in the endoplasmic reticulum, resulting in a severe reduction at the plasma membrane (4–6). When expressed in Xenopus oocytes (where the transport block can be overcome), the G480C protein has an apical plasma membrane Cl– channel activity identical to that of wild-type CFTR (6). Contradictory reports exist for F508 function at the apical membrane, some suggesting reduced function and others suggesting normal function (7–9).
We created mutant mice that carry the G480C mutation by gene targeting using the ‘hit and run’ technique (10,11). These mice are comparable to the Cftrtm1Eur mice where the ‘hit and run’ technique was used to introduce the F508 mutation into the mouse (12). In both these Cftr mutant mice the only alteration in the Cftr gene is the introduced mutation, and transcription of the mutant allele does not deviate from wild-type levels (13). This is in contrast to replacement mouse models, where the gene targeting event introduces plasmid sequences into an intron and the transcription of the mutant allele is reduced (14,15). Thus, for example, in the Cftrtm2Cam F508 mice the level of Cftr mRNA is 15% of wild-type levels (14). In such mutant mice (e.g. the F508 Cftrtm2Cam and the G551D Cftrtm1G551D mice), it is therefore difficult to assess whether the phenotype is due to the precise mutation or principally related to a reduction in the level of the transcript.
The Cftrtm1Eur mice, where F508 Cftr transcript levels are normal, possess a small, residual forskolin response in the gut (12), not evident in the intestine of the ‘null’ or other F508 mutant mice. In the nasal tissue these F508 mutant mice cannot be distinguished from littermates on the basis of their low chloride response (12). The ‘null’ and low level transcript F508 mice die perinatally or at weaning due to intestinal blockage but do display a clear defect in their nasal bioelectrics. In this study, an accurate mouse model of the G480C mutation was used to assess the phenotype of another ‘severe’ CF mutation in vivo and to clarify the organ-specific consequences of a mistrafficking mutant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Cftrtm2Hgu mice which carry the G480C mutation
ES cells modified at the Cftr locus to possess the G480C mutation in exon 10 by ‘hit and run’ gene targeting have previously been described by Dickinson et al. (11). Four ES cell clones modified to possess the G480C mutation which had normal karyotypes were used for blastocyst injections. Germline transmission of the mutation was detected by genotyping agouti offspring from mating the chimeric males to C57Bl/6 females and was observed in two independent clones, with all mice used in this study originating from a single transmitting chimeric founder animal. Homozygous G480C mutant mice are designated Cftrtm2Hgu following the Mouse Nomenclature Committee guidelines. A novel NsiI restriction enzyme site was created at the site of the G480C mutation (Fig. 1A) and restriction enzyme digestion verified the faithful replacement of the wild-type exon 10 with the mutant exon 10 (Fig. 1B–D). PCR amplification of exon 10 and digestion with NsiI differentiated the enzyme-resistant wild-type band from the G480C-containing band, which can be digested with the enzyme (Fig. 1E).
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HindIII molecular weight marker. (D) Production of homozygous Cftrtm2Hgu/tm2Hgu mice. Heterozygote Cftrtm2Hgu/+ mice were intercrossed and litters genotyped by Southern blot analysis of NsiI digests probed with 1.3XH. G, Cftrtm2Hgu/tm2Hgu mice; H, heterozygous Cftrtm2Hgu/+ mice; W, wild-type Cftr+/+ mice; M, 
X174 HaeIII molecular weight marker.G480C mutant mice express the mutant allele at wild-type levels
Both male and female mice heterozygous for the G480C allele were included in the study and a range of tissues investigated. The
F508 mice generated by replacement gene targeting (Cftrtm2Cam) show differences in the extent of allele bias between tissues (14). Cftr mRNA expression was demonstrated by RT–PCR between exons 9 and 10 and was observed in all of the tissues investigated (lung, colon, jejunum, ileum and testis). Samples were taken during the linear range of the PCR amplification as determined by real-time luminescence assay (Materials and Methods), to reduce heteroduplex formation. G480C products were distinguished from wild-type by the presence of the novel NsiI restriction site polymorphism engineered adjacent to the missense change. Densitometric quantitation of PCR products after NsiI digestion indicated approximately equal ratios of each allele in the ileum, jejunum, testis and lungs (Fig. 2 and Table 1), except in one lung sample which appeared to have undergone degradation following digestion. These results indicate that the modified allele was expressed at the same level as the wild-type allele and therefore the presence of the G480C mutation has no effect on the level of expression.
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G480C CFTR is incompletely processed
Analysis of CFTR processing in isolated jejunal enterocytes of wild-type mice by western blotting demonstrated a normal pattern of CFTR isoforms with the core-glycosylated isoform of CFTR (Fig. 3, band B) in the ER, and the mature, fully-glycosylated isoform (Fig. 3, band C) in the plasma membrane. Band B was identified only in the crude membrane preparation (Fig. 3, lane 1) consisting of a mixture of ER, Golgi and plasma membranes, whereas band C was additionally detected (and
2-fold enriched) in the brush border membrane vesicle (BBMV) preparations (Fig. 3, lane 3) consisting of virtually pure apical membranes. In contrast, intestinal epithelium from homozygous G480C mice showed a normal intensity of the B band of CFTR in the crude jejunal membrane fraction (Fig. 3, lane 2), but a strongly reduced intensity of the C band [measured as 8% (±2), n = 4, residual CFTR compared to wild-type as determined by dilution-calibrated scanning of the bioluminescence] in the BBMV (Fig. 3, lanes 2 and 4). This outcome clearly demonstrates that the G480C CFTR mutant protein is retained in the ER of the enterocytes in vivo and that only a very small fraction is able to escape the quality control mechanism in the ER and reach the cell surface.
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Immunocytochemical detection of CFTR in the jejunum from wild-type mice using the R3195 antibody [in keeping with previous findings (13)], showed intensive staining of the apical region of all crypts (Fig. 4A). In addition, due to the improved sensitivity of CFTR staining in microwave-treated paraffin sections as compared to cryosections, CFTR immunoreactivity was also prominent in the brush border membrane of lower- and mid-villus cells. In contrast, CFTR expression in crypts and villi from homozygous G480C mutant mice remained below the detection level of the immunocytochemical technique (Fig. 4B). This finding confirms the results of the western blotting shown in Figure 3 and is in line with the concept of a processing defect affecting the maturation of G480C CFTR in both the crypt and villus compartments.
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Phenotype of the CF mutant mice homozygous for the G480C mutation
Figure 5 demonstrates that genotypes of the litters produced from matings between G480C heterozygous mice did not deviate from the expected Mendelian ratio of wild-type:heterozygotes:homozygotes of 1:2:1, and no reduction in the number of homozygotes was observed. This was true in the offspring from mating heterozygotes from the outbred 129/Ola/C57Bl/6N background and also from mating heterozygotes from a fourth generation of backcross matings to C57Bl/6N mice. Furthermore, homozygous G480C mice did not show any increased mortality over wild-type animals (pre- or post-weaning) over an 18 month period. The weight (at weaning) of the three genotypes did not differ significantly (Fig. 5), and both males and females were fertile (data not shown).
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Histological analysis of intestinal sections demonstrated focal hypertrophy of goblet cells in the G480C homozygous mutant mice. Sections from the G480C mutants could easily be distinguished from wild-type by this criterion alone (Fig. 4). However, this hypertrophy is much less severe than that observed in the ‘null’ [Cftrtm1Unc or Cftrtm1Cam (16,17)] mice and is similar to that observed in the Cftrtm1Eur
F508 mice (12). In addition, we saw no evidence (n = 20 mice) of dramatic distension of crypts in the small intestine, or blockage, nor any evidence of an abnormal coiled, worm-like caecum impacted with sticky fecal material which is described as the only obvious intestinal abnormality in the Cftrtm1Unc mice killed while apparently still healthy (16).
The Cftrtm1Unc ‘null’ mice and Cftrtm1Eur F508 ‘hit and run’ mice display white teeth as a result of decreased enamel mineral content and increased magnesium (18; H.R.de Jonge, unpublished data). Both these strains of mice can be genotyped purely on the basis of their white tooth colour. However, the incisor teeth of the G480C mutant mice were not abnormally white (data not shown).
Electrophysiological characteristics of G480C CFTR mice
The reduced chloride permeability of the epithelium due to CFTR dysfunction, causes typical abnormalities in the ion transport of different epithelia in both CF individuals and Cftr mutant mice. The G480C CF mice do not suffer from the intestinal blockage (the first signs of which are located in the caecum) that is seen in mice with a complete disruption of Cftr expression, so we examined the electrophysiological profile of these animals in the intestine.
In the caecum, Ussing chamber measurements revealed that the initial baseline Isc (short circuit current) was significantly (P = 0.0001) reduced in the G480C homozygous mutants compared to controls (Fig. 6A). However, the response to forskolin, which activates CFTR through an increase in cAMP, was not significantly different between wild-type and mutant animals. Carbachol (which stimulates Ca2+-mediated chloride transport), probably also via CFTR in the intestine, was, however, significantly (P = 0.01) reduced compared to wild-types. However, in the jejunum (Fig. 6B), the baseline Isc was not significantly different between the mutants and wild-types, but the forskolin response was significantly (P 
0.01) different. The carbachol response, as in the caecum, was significantly reduced compared to littermates (P < 0.01).
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Several studies have demonstrated that in the intestine of rodents, the baseline and stimulated secretions are independent when the stimulation is made with forskolin. Naturally, cAMP levels may be elevated via prostaglandin increase. However, these studies show that the forskolin- and prostaglandin-mediated pathways are independent in as far as normal forskolin-mediated secretion can be achieved following prostaglandin-mediated secretion (19–21). In addition, we can demonstrate (H.R.de Jonge, unpublished data) that the basal short circuit current in the murine intestine was not affected by 10–5 M indomethacin (a potent inhibitor of prostaglandin synthesis) and is therefore not prostaglandin/cAMP mediated. Therefore, we are confident that the responses generated in this study by forskolin are independent of any prostaglandin-mediated baseline secretion in the jejunum and caecum.
We also examined the nasal biolelectrics of these mice (Fig. 6C) and in common with all other described CFTR mutant mice (D.J.Davidson and J.R.Dorin, The CF mouse: an important tool for studying cystic fibrosis. Exp. Rev. Mol. Med., http://www-ermm.cbcu.cam.ac.uk/01002551h.htm), the baseline was significantly (P 0.001) raised. The response to a low chloride gradient was significantly (P = 0.0001) reduced in the G480C mutant animals, in contrast to the normal response reported in the Cftrtm1Eur F508 mouse.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The mutant mice we present here mimic human CF individuals with the ‘severe’ G480C mutation. The G480C mutant protein was detected in a pancreatic insufficient African–American CF patient (6). Western blot analysis and immunofluorescence analysis revealed that even when overexpressed in 293T cells, no fully glycosylated nor apically localized protein was detectable. This suggested that the G480C protein was similar to the
F508 protein and subject to defective intracellular processing. We demonstrate that when replicated in the mouse, the G480C mutant CFTR is mislocalized and the defect in chloride ion transport characteristic of CF varies between tissues and is present in the nose and jejunum but absent from the caecum with no evidence of fatal gut blockage.
G480C mutant mice express normal levels of the mutant allele
The ‘hit and run’ procedure used to generate these mice results in the only genomic alteration being at the site of the mutation. It follows that the message levels of the mutant allele will be equivalent to the wild-type allele. In F508/wild-type heterozygous humans, mRNA of both alleles is present at an equal ratio (22). Mice created by gene targeting for the common F508 mutation by replacement gene targeting (14), used a vector which included a reverse orientation HPRT mini-gene selection cassette in intron 10 along with the F508 mutation in exon 10. The derived heterozygote mice were determined to have a Cftr expression bias at the mRNA level in favor of the wild-type allele over the engineered F508 allele. No reasons for this bias were investigated or considered in the original paper, but the most likely possibility is interference from the transcription of HPRT in the reverse orientation, from within intron 10, although binding of transcription repressors, chromatin modification, methylation and RNA processing differences are all possibilities. These Cftrtm2Cam F508 mutant mice have a very low level of mutant transcript and a phenotype very similar to the ‘null’ Cftr mutant mice where the vast majority die perinatally or around weaning from intestinal blockage. The Cftrtm1Kth F508 mice were also made by replacement gene targeting and again have a high level of early death from intestinal blockage (23). The ‘hit and run’ (Cftrtm1Eur) F508 mutant mouse, in contrast, has a normal level of mutant transcript and although it displays evidence of growth retardation at weaning (12), it does not demonstrate a phenotype of death from gut blockage. In contrast to the F508 mice generated by replacement gene targeting, the Cftrtm1Eu F508 ‘hit and run’ mice and the Cftrtm2Hgu G480C ‘hit and run’ mice generated here both express normal levels of the mutant allele.
G480C mutant protein is mislocalized
The majority of G480C CFTR when subjected to western blot analysis is clearly mislocalized in vivo in the mouse intestine. However, a low level (8% of wild-type) of mature band C is evident in the BBMV preparations. This outcome clearly suggests that the majority of G480C CFTR mutant protein is retained in the ER of the enterocytes in vivo but that a significant fraction is able to escape the quality control mechanism in the ER and travel to the cell surface. In jejunal BBMV samples from F508 Cftrtm1Eur mice the residual level of mature protein was estimated at 3% (±1) of wild-type (H.R.deJonge and R.Willemsen, unpublished data). This suggests that the G480C processing defect in the intestine is slightly less severe than that of the F508 mutant in vivo. It should be noted that although 8% of normal levels of mature G480C was detectable in western blot analysis of BBMV, only cytoplasmically localized protein could be detected by immunohistochemistry.
The phenotype of the Cftrtm2Hgu G480C mutant mice is mild
The Cftrtm2Hgu G480C mutant mice do not demonstrate a phenotype of death from gut blockage and unlike the F508 Cftrtm1Eur mice do not even display any evidence of growth retardation at weaning. The histology of the G480C intestine is not severely abnormal unlike the Cftrtm1Unc ‘null’ mice, which display extensive goblet cell hyperproliferation, increased mucus accumulation and luminal obstruction. The F508 mice display focal hypertrophy of goblet cells in the crypts of the small intestine. The G480C mice (Fig. 4), also do not have any gross abnormalities but do display a mild focal hypertrophy of goblet cells comparable to the data reported for the F508 Cftrtm1Eur homozygotes.
The classic CF chloride transport defect is not present in the caecum and may account for the lack of intestinal blockage
The G480C mutant mice do not show a defect in their forskolin response in the caecum, although baseline and carbachol response are altered compared to wild-type. The F508 ‘hit and run’ mutant mice, in contrast to the G480C mice, have a significant but markedly reduced (by 85%) forskolin-activated chloride ion conductance in the caecum compared to wild-type (13). In fact, all previously reported CF mutant mice that show a reduced incidence of death from intestinal obstruction (Cftrtm1Hgu, Cftrtm1Eur, Cftrtm1G551D), also show an increased caecal cAMP response compared to Cftr mutant mice with a high death rate from intestinal blockage (http://www-ermm.cbcu.cam.ac.uk/0100255lh.htm). The caecum is the region of the gut where most of the intestinal blockage occurs in ‘null’ Cftr mice, so a normal forskolin response (but not baseline or Ca2+ agonist response) in this tissue is likely to be important in ameliorating this phenotype.
Variation between the bioelectric phenotypes of F508 and G480C ‘hit and run’ mutant mice could be explained by the effect of modifier genes of residual chloride secretion present in the genetic backgrounds on which the mutations have been bred. This is unlikely because French et al. (13) and ourselves (data not shown) have demonstrated that the electrophysiological profiles in the caecum, ileum and jejunum of wild-type 129 and C57Bl/6 mouse strains are not significantly different. In addition, neither appear to have any dominant gene effects on the intestinal bioelectrics. Finally, the G480C mice show 100% survival on a mixed 129/C57Bl/6J background (the same as that reported for the Cftrtm1Eur mice with 100% survival) and this does not alter after four backcrosses onto the C57Bl/6J background. This is the same genetic background as that reported for the Cftrtm1Unc ‘null’ mice with only 20% survival, and reproduced in our animal house (15).
It is probable that the normal forskolin response in the G480C mice compared to the abnormal response in Cftrtm1Eur F508 mice is due to slightly more G480C (8% versus 3%) being correctly processed and reaching the apical membrane. However, the results from Xenopus oocyte experiments using human CFTR mRNA, strongly suggested that this observed difference between the G480C and F508 response to forskolin was consistent with a trafficking/processing defect in G480C CFTR, and an additional conductance defect in F508 CFTR (6). We doubt whether this additional conductance defect is the case here as the F508 ‘hit and run’ mice have identical channel characteristics to wild-type in the gall bladder epithelia (13).
The fact that ‘hit and run’ F508 mutant mice (Cftrtm1Eur) display runting but no evidence of the fatal intestinal blockage presented by ‘null’ CF mice is in contrast to F508 homozygous patients, who do not have reduced incidence of meconium ileus when compared to patients with ‘null’ mutations. One possible explanation is that the G480C and F508 mouse phenotypes appear to be different to ‘null’ mice because of differences in human/mouse physiology and gut architecture, and the mouse is more sensitive to small increases in CFTR function.
Electrophysiological phenotype of the murine G480C mutant protein varies between tissues
An unexpected finding was the organ-specific differences in CFTR-related electrophysiology in the mutant mice. As discussed above, we suggest that a normal cAMP-mediated chloride secretion in the caecum is due to 8% correctly localized CFTR. This agrees with our previous work (24), where we demonstrate, using intercrossed mutants, that a very low level of functional CFTR (5%) is sufficient to have a disproportionate effect on chloride ion transport and a major effect on phenotype (survival due to lack of intestinal obstruction). Interestingly, the caecal baseline and Ca2+-activated chloride response remain abnormal despite a normal forskolin response. This implies that these aspects may not be key markers of intestinal blockage and, in addition, that there is a CFTR dose response in terms of restoration of function within an organ. These are important considerations when monitoring the efficacy of therapies aimed at amelioration of the phenotypes in CF individuals.
In the nose of the Cftrtm2Hgu mice, in common with all other reported CF mice and individuals (http://www-ermm.cbcu.cam.ac.uk/01002551h.htm), the baseline potential difference (PD) is raised, indicating a defect in sodium absorption. The familiar defect in cAMP-mediated chloride secretion was also observed in the nose, which is in direct contrast to the Cftrtm1Eur F508 mutant mouse but in common with all other mutant mice including the 10% residual function mouse (Cftrtm1Hgu). So, the fact that the cAMP-stimulated response is not defective in the caecum, but the nasal low chloride response is defective, again demonstrates inter organ differences in this ‘dose response’. This must reflect either tissue-specific alterations in the level of mature G480C CFTR with organ-specific subtle translational/post-translational differences, or compensatory pathways altering the bioelectric phenotype. We tentatively suggest that correction may be more difficult in the airways as compared to the large intestine.
The Cftrtm2Hgu G480C mutant mouse is a valuable tool for therapy testing
Both the defects in sodium absorption and in chloride secretion are evident in the nose of the G480C mutant mouse and this is widely held to be the mouse tissue that mimics the human respiratory tract phenotype most closely (25). Seventy percent of CF chromosomes have the F508 mutation and so the vast majority of CF individuals possess at least one F508 allele. This makes pharmacological strategies aimed at improving the function of the F508 mutant CFTR highly relevant. The three mouse models generated to date that express the F508 mutant allele either suffer a high level of intestinal blockage and death (Cftrtm2Cam, Cftrtm1Kth), or survive well but do not have the bioelectric defect of chloride ion transport in the respiratory tract. The fact that this G480C mutant mouse combines a mistrafficked CFTR mutation (similar to the F508 CFTR) with normal survival means that it is an excellent in vivo model for testing drugs aimed at mutant CFTR relocation strategies.
In conclusion, the introduction of the G480C mutation into the mouse Cftr gene, using the ‘hit and run’ technique mimics the human allele with normal levels of Cftr mRNA production. This has allowed us to demonstrate that the majority of the G480C mutant protein is mislocalized, but a low level of mature CFTR is detectable by immunoblot. The phenotype of the mutant animals is extremely mild, and does not include severe gut blockage or growth retardation. The G480C homozygous mutant protein has different ion transport effects in different organs with pronounced effects on the baseline in the nose and the caecum. In the nose, the mutant animals have increased absorption whilst in the caecum, reduced secretion is evident. Reduced stimulation of chloride secretion has been found in the nose and the jejunum but a normal response was found in the caecum and this is most likely responsible for the lack of fatal intestinal blockage and the normal weight of the G480C mutant mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene targeting in ES cells
Targeting methodology is as described previously by Dickinson et al. (11). Screening of litters for transmission of the G480C allele was performed by PCR using 25 base pair primers used to amplify exon 10 from positions 1530 to 1720 in the Cftr gene, described previously by Dickinson et al. (26), followed by NsiI digestion.
Real-time RT–PCR analysis
Cftrtm2Hgu/+ heterozygous G480C mice were killed by CO2 asphyxiation. Tissues were dissected out, washed in PBS and snap frozen in liquid nitrogen. Samples were thawed, homogenized in 1 ml RNAzol B (Biogenesis) and 100 µl chloroform added for phase extraction. Nucleic acids were ethanol precipitated at 4°C, washed with 80% (v/v) ethanol and resuspended in 200 µl DEPC-treated dH2O for 18 h at 4°C. The yield was determined spectrophotomerically after heat denaturation of 1/250 sample dilutions. An OD260/280 ratio of 2 was typically obtained. One microgram of total cellular RNA was reverse transcribed in 20 µl total volume reactions containing AMV RTase (Boehringer Mannheim) and the manufacturer’s recommended concentrations of buffer, Mg2+, dNTP, gelatin and RNase inhibitor. Random hexanucleotides were used to prime synthesis. In addition to the manufacturer’s recommendations, a 10 min room temperature incubation was included to allow primer annealing, and reverse transcription was carried out at 42°C for 1 h. The enzyme was heat killed by incubation at 95°C for 15 min and samples stored at –70°C. For each reverse transcription reaction carried out, a reverse trancriptase negative control was also performed.
Measurements of laser-activated fluorescence of nucleic acid intercalated SYBR green (FMC Bioproducts) were used to define a reproducible window in which PCR products were sufficiently abundant to be detected by Southern blot, but still within the exponential range of amplification. Reactions were carried out in an Idaho Technologies ‘Light Cycler’. Thermal cycling proceeded through 95°C for 0 s (subsequent to a 30 s initial denaturation), 57°C for 0 s and 74°C for 6 s, the rapid ramping of temperature achievable through the use of glass capillary tubes as reaction vessels. Cycling was performed until the target window of amplification was attained. At this point the temperature was held at 74°C at the end of the 6 s normal extension to allow removal of samples which were slowly cooled to room temperature. Semi-quantitative RT–PCR and appropriate controls were amplified using primers M9AI (5'-AGCAATGGTGACAGAAAACATTCC-3') and M10B (5'-CTGCTGTAGTTGGCAAGCTTTGAC-3'). Reagents were as recommended (Biogene). AmpliTaq (Perkin Elmer) was the polymerase used. PCR products were electrophoretically separated at 100 V on 3% (w/v) agarose gels after digestion to completion with NsiI. Gels were capillary blotted onto nylon transfer membrane (MSI), baked at 80°C for 1 h and UV crosslinked with a Stratalinker (Stratagene). Hybridization of the 32P end-labelled 24mer oligonucleotide M10BI (5'-TCATCATAGGAAACACCAAAGATG-3') was performed overnight at 50°C and membranes washed in 4x SSC at 50°C. Densitometric quantitation of signals was determined by a phosphoimager and using the Image Quant software area integration algorithm (Molecular Dynamics).
Western blot analysis
Wild-type mice and littermate mice (backcrossed for four generations onto the C57Bl/6 strain background) carrying the G480C mutation were anaesthesized 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 from two mice 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 (Boehringer Mannheim), 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 (27) 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 van Dommelen et al. (27). 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 slabgels 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).
CFTR antibody
The rabbit polyclonal antibody R3195 (kindly donated by Dr Christopher R.Marino, University of Tennessee, Memphis, TN) 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 (13).
Immunocytochemical analysis
Wild-type mice and littermate mice carrying the G480C mutation were killed by cervical dislocation, the intestine was dissected and the jejunum was rinsed with ice-cold saline and fixed in 3% (w/v) paraformaldehyde for 16 h, prior to standard paraffin embedding. Sections (5 µm) were deparaffinized, followed by microwave treatment in 0.01 M sodium citrate solution according to Devys et al. (28). Endogenous peroxidase activity was blocked by a 30 min preincubation 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 h followed by a 45 min incubation with a peroxidase-conjugated secondary antibody. Enzymic 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 or without primary and secondary antibody. In both cases, background labelling appeared negligible.
Electrophysiological analysis
G480C homozygous animals were assessed in vivo (nose) and in vitro (jejunum, caecum) and compared with littermate controls.
Jejunum and caecum. These tissues were mounted in standard gas-lift 0.28 cm2 Ussing chambers at 37°C under short circuit conditions. Following a 30 min equilibration period, the baseline current and transepithelial PD were measured and the conductance calculated. Subsequently, amiloride (10 µM, mucosally) was added followed 5 min later by forskolin (10 µM, serosally). Carbachol (1 mM, bilaterally) was administered at the plateau of the forskolin response and the chloride component used to assess muscarinic-dependent chloride secretion. Thus, in the presence of amiloride, both cAMP-dependent and calcium-dependent chloride secretion were examined.
Nasal. Baseline potential difference and responses to perfused HEPES Krebs low chloride buffer (pH 7.4) in the presence of amiloride (100 µM) were measured as described previously by Smith et al. (29). Briefly, a fluid-filled dual channel catheter was placed 5 mm within the nasal cavity. The measuring channel and the reference line were connected via calomel electrodes to a hand-held computer (Psion) that provided a simultaneous visual chart presentation (Logan Research Ltd).
Statistics. Values are expressed as mean ± SEM for convenience. The Mann–Whitney U-test was used to compare groups and the null hypothesis rejected at P < 0.05.
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ACKNOWLEDGEMENTS |
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The authors would like to thank Lies-Anne Severijnen and Alice Bot for technical assistance, and Chris Marino for the gift of antibody R3195. The study was supported by the UK Cystic Fibrosis Research Trust, MRC and a Wellcome Trust Senior Clinical Fellowship (E.W.F.W.A.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 131 467 8410; Fax: +44 131 343 2620; Email: julia@hgu.mrc.ac.uk Present addresses: Paul Dickinson, Centre for Inflammation Research, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK Martin S. Taylor and David J. Porteous, Medical Genetics Section, Department of Medical Sciences, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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