ABCMdb

ABCC7 p.Ser489*

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Publications

PMID: 10777364 [PubMed] Wang J et al: "A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients."

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570 SYLVAIN R RIVARD* CHRISTIAN ALLARD† JEAN-PIERRE LEBLANC† MARCEL MILOT† GERVAIS AUBIN† FERNAND SIMARD† CLAUDE FÉREC‡ MARC DE BRAEKELEER†§¶ *Département des Sciences Fondamentales, Université du Québec à Chicoutimi, Canada Table 1 Distribution of cystic fibrosis patients diagnosed before the age of 5 by age groups in Saguenay-Lac-Saint-Jean, (A) by genotype, (B) by mutation 0-10 years 10.1-20 years Over 20 years All ages No % No % No % No % (A) Genotype F508/ F508 15 (1) 40.5 21 (2) 36.2 18 (3) 42.9 54 (6) 39.4 F508/621+1G→T 12 (1) 32.4 16 (1) 27.6 10 (1*) 23.8 38 (3*) 27.7 F508/A455E 1 2.7 6 10.3 5 11.9 12 8.8 F508/I148T 1 2.7 1 1.7 2 1.5 F508/Y1092X 3 (1) 5.2 1 2.4 4 (1) 2.9 F508/Q890X 1 2.4 1 0.7 F508/R1158X 1 2.4 1 0.7 621+1G→T/621+1G→T 2 (1) 5.4 4 6.9 1 2.4 7 (1) 5.1 621+1G→T/A455E 1 2.7 4 6.9 3 7.1 8 5.8 621+1G→T/711+1G→T 2 (1) 5.4 2 (1) 3.4 4 (2) 2.9 621+1G→T/Y1092X 1 2.7 1 0.7 621+1G→T/S489X 1 2.7 1 0.7 621+1G→T/G85E 1 (1) 1.7 1 (1) 2.4 2 (2) 1.5 A455E/R117C 1 2.7 1 0.7 N1303K/I148T 1 2.4 1 0.7 Total 37 58 42 137 Death (4) 10.8 (6) 10.3 (5*) 11.9 (15*) 10.9 (B) Mutation F508 16 (1) 43.2 25 (3) 43.1 21 (3) 51.2 62 (7) 45.6 621+1G→T 18 (3) 48.6 23 (3) 39.7 12 (2*) 29.3 53 (8*) 39.0 A455E 3 8.1 10 17.2 8 19.5 21 15.4 Total 37 58 41 136 Death (4) 10.8 (6) 10.3 (5*) (12.2) (15*) (11.0) ( ): Number of deaths. Login to comment

PMID: 10777368 [PubMed] Rivard SR et al: "Correlation between mutations and age in cystic fibrosis in a French Canadian population."

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PMID: 10926557 [PubMed] Larson JE et al: "CFTR modulates lung secretory cell proliferation and differentiation."

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54 METHODS Colony Maintenance The S489X cftr-mutant UNC mouse strain was obtained from Jackson Laboratories as a fifth-generation backcross. Login to comment
57 Under these conditions, the S489X cftr-mutant mice develop intestinal obstruction, and Ͻ5% survive into adulthood. Login to comment
72 Wild-type CFTR and the knockout S489X allele were amplified in parallel reactions with conditions provided by Jackson Laboratories. Login to comment

PMID: 10930443 [PubMed] Kelley TJ et al: "In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium."

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324 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

PMID: 10970820 [PubMed] Rajan S et al: "Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors."

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84 Immunohistochemistry Mice homozygous for the CFTR S489X (null) mutation or wild-type littermate controls (cftr -/- and ϩ/ϩ) were inoculated with P. aeruginosa impregnated in agar beads as previously described (10). Login to comment

PMID: 11029305 [PubMed] Thomas EJ et al: "Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines."

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42 By use of this protocol, the CFTR knockout (S489X) gave a 210-bp product and the wild-type CFTR yielded a 190-bp product. PCR products were identified by electrophoresis on 2% (wt/vol) and 3% (wt/vol) agarose gels for TAg and CFTR products, respectively. Login to comment
90 The CFTR and TAg genotype of each cell line was confirmed by PCR, which indicated that MTE18- (-/-) cells were homozygous for the S489X CFTR knockout, MTE7b-(ϩ/-) cells were heterozygous for this mutation (Fig. 1E), and both cell lines were positive for TAg (Fig. 1F). Login to comment
155 In the present study, two such cell lines have been generated: one from a mouse homozygous for the S489X CFTR knockout and one from a mouse heterozygous for this mutation. Login to comment

PMID: 11121394 [PubMed] Takacs-Jarrett M et al: "Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene."

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41 The goal of this work was to cross the ImmortoMouse (26) with the University of North Carolina (UNC) CF knockout mouse (CFTR S489X) (40) and develop genetically well-matched, conditionally immortalized CF and non-CF epithelial cell lines. Login to comment
42 METHODS Animals Male mice, homozygous for a temperature-sensitive SV40 large T antigen transgene (ImmortoMouse; CBA/ca X C57B1/10 strain; Charles River Laboratories) (26), were bred with female mice that were heterozygous for the S489X CFTR mutation (UNC; CFTR ϩ/-; C57BL/6J X F129 strain) (40). Login to comment
50 Primers 5 and 6 were used to amplify the S489X neodisrupted allele of CFTR (NEO). Login to comment
145 The CF cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) were negative for the wild-type CFTR allele (faint 200-bp bands are non-CFTR PCR products) and positive for the S489X neodisrupted allele of CFTR. Login to comment
151 Magnification, ϫ270. both the wild-type CFTR allele and the S489X neodisrupted allele, whereas one of the non-CF cell lines (mSEC1) was positive for the wild-type CFTR allele and negative for the S489X neodisrupted allele of CFTR. Login to comment
179 Wild-type cystic fibrosis transmembrane conductance regulator (CFTR), neomycin disrupted S489X CFTR (NEO), and Immorto (IM) alleles were identified from PCR products separated by gel electrophoresis. Login to comment

PMID: 11274233 [PubMed] Nakanishi K et al: "Role of CFTR in autosomal recessive polycystic kidney disease."

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38 The absence of functional CFTR in the animal model for CF (CFTR S489X or UNC) used in this study has been demonstrated extensively by Western analysis, ribonuclease protection assay, electrophysiologic analysis, and PCR analysis. Login to comment

PMID: 11350833 [PubMed] Mhanna MJ et al: "Nitric oxide deficiency contributes to impairment of airway relaxation in cystic fibrosis mice."

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23 To test this hypothesis, we compared the airway relaxant response of CF mice homozygous for the null S489X mutation with that of their normal littermates. Login to comment
28 Materials and Methods Animals Male and female mice homozygous for the S489X (CFTR-/- ) mutation of the CFTR and their normal littermates (CFTRϩ/ϩ ) were studied (18, 19). Login to comment
35 of Pediatrics, Washington University School of Medicine, St. Louis, MO. Abbreviations: analysis of variance, ANOVA; cystic fibrosis, CF; CF transmembrane conductance regulator, CFTR; mice homozygous for the S489X mutation of the CFTR, CFTR-/- mice; normal littermates of CFTR-/- mice, CFTRϩ/ϩ mice; electrical field stimulation, EFS; inducible NOS, iNOS; NG-nitro-L-arginine methylester, L-NAME; neuronal NOS, nNOS; nitric oxide, NO; NO synthase, NOS; prostaglandin, PG; standard error of the mean, SEM; substance P, SP; percentage of maximal tracheal tension, %T max. Login to comment

PMID: 11390493 [PubMed] Schroeder TH et al: "Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract."

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5 We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the ⌬F508 Cftr allele or an allele with a Cftr stop codon (S489X). Login to comment
6 Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 ⌬F508 Cftr mice, whereas ⌬F508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. Login to comment
47 Transgenic CF mice included the following strains: C57BL/6 mice homozygous for the ⌬F508 allele of mouse Cftr (B6.129S6-Cftrttm1Kth ) (10), noninbred homozygous ⌬F508 Cftr mice (11), and doubly transgenic S489X Cftr-fatty acid binding protein (FABP)huCftr mice (no murine Cftr protein but expressing wild-type human (hu) CFTR protein in the gastrointestinal tract from the huCFTR gene under the control of the mouse FABP promoter) (12). Login to comment
48 The doubly transgenic S489X Cftr-FABPhuCftr mice were bred as homozygotes. Login to comment
113 Plating serial dilutions of the gastrointestinal organs and the nasopharyngeal anatomic area of intranasally infected S489X-FABPhuCftr mice revealed that the applied bacteria were not swallowed but remained in the nasopharynx of these transgenic CF mice (data not shown). Login to comment
122 We first compared the total and internalized amount of bacteria in the lungs 4.5 h after tracheal infection of anesthetized wild-type C57BL/6 mice with that in two strains of transgenic CF mice: noninbred S489X FABPhuCftr mice and inbred B6.129S6-Cftrttm1Kth . Login to comment
123 We found that lung cells of wild-type C57BL/6 mice internalized 7.6 times more P. aeruginosa PAO1 bacteria than did the C57BL/6 mice homozygous for the ⌬F508 Cftr allele (B6.129S6-Cftrttm1Kth mice) and 3.9 times more bacteria than did the S489X-FABPhuCftr mice ( p Ͻ 0.001, ANOVA and Fisher PLSD; Fig. 1A). Login to comment
125 Coincident with the impaired internalization of P. aeruginosa in the respiratory tract of the two transgenic CF mouse strains, the total level of the infecting bacteria measured in the lungs was increased 22-fold in homozygous ⌬F508 Cftr C57BL/6 mice and 30-fold in S489X-FABPhuCftr mice, whereas in wild-type mice the increase in the bacterial burden over the inoculating dose was only 10-fold (Fig. 1B; both CF strains p Ͻ 0.01, ANOVA and Fisher PLSD compared with wild-type mice). Login to comment
134 Chroneos et al. (7) recently reported that there was no difference in clearance of P. aeruginosa from the lungs of S489X-FABPhuCftr mice compared with wild-type mice. Login to comment
198 Chroneos et al. (7) recently reported no difference in clearance of P. aeruginosa strain FRD-1 from the lungs of S489X-FABPhuCftr mice in comparison to wild-type mice, 24 h after a dose of 1.5 ϫ 107 CFU delivered intratracheally. Login to comment
204 Chroneos et al. (7) also studied strain PAO1, as we did, but they only compared clearance between wild-type mice and double-transgenic S489X-FABPhuCftr mice that had been further engineered to overexpress huCFTR in the lungs under the control of the surfactant protein C (SP-C) promoter (SP-ChuCFTR mice). Login to comment
208 For strain PAO1, Chroneos et al. (7) did not compare clearance in the transgenic S489X-FABPhuCFTR mice lacking CFTR in the lung with wild-type mice. Login to comment
263 This occurred in both inbred and outbred transgenic mouse lines homozygous for the ⌬F508 allele of Cftr and in S489X Cftr-FABPhuCftr mice, which have no intact CFTR protein. Login to comment

PMID: 11404242 [PubMed] Velsor LW et al: "Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice."

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42 The CFTR-KO mice used in this study were congenic B6.129P2-Cftrtm1Unc , which possess the S489X mutation in CFTR that renders the CFTR protein nonfunctional (9, 40). Login to comment
44 In this study, the CFTR-KO mice, homozygous for the S489X mutation, were compared with their WT littermates. Login to comment

PMID: 11447196 [PubMed] Sajjan U et al: "Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice."

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310 In contrast, when another strain of CFTR-deficient mice (Cftrm1UNC ; S489X null mutant) was challenged with S. aureus, no differences in bacterial clearance between CFTR-deficient mice and the controls were demonstrated (55). Login to comment

PMID: 11694004 [PubMed] Gawenis LR et al: "Mineral content of calcified tissues in cystic fibrosis mice."

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36 MATERIALS AND METHODS Animals This study was performed on CF mice with the S489X mutation (cftrtm1Unc) (14) and ∆F508 mutation (cftrtm1kth) (15). Login to comment

PMID: 11741811 [PubMed] Cobb BR et al: "A(2) adenosine receptors regulate CFTR through PKA and PLA(2)."

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108 The cftr(-/-) mice carried two copies of the human cftr cDNA, which contains a stop codon at position 489 (S489X). Login to comment

PMID: 11823525 [PubMed] Soltys J et al: "Functional IL-10 deficiency in the lung of cystic fibrosis (cftr(-/-)) and IL-10 knockout mice causes increased expression and function of B7 costimulatory molecules on alveolar macrophages."

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43 Mice homozygous for the S489X (B6.129P2-Cftrtm1Unc ) mutation of the CFTR gene, congenic (n ϭ 10 generations) onto C57BL/6J background, (18) and their normal littermates, designated as CFTR-/- and CFTRϩ/ϩ , respectively, were bred in our Animal Core facility. Login to comment

PMID: 11945068 [PubMed] Ziady AG et al: "Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor."

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172 We studied 8to 10-week-old B6.129P2-cftrtm1Unc mice (CF knockout, S489X mice) [25] fed a liquid diet [26], or STOCK Cftrtm1Unc - TgN(FABPCFTR)#Jaw mice [27], which are CF mutant mice with the defect corrected only in intestinal epithelium by transgenic human CFTR. Login to comment

PMID: 11960971 [PubMed] Freedman SD et al: "Characterization of LPS-induced lung inflammation in cftr-/- mice and the effect of docosahexaenoic acid."

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28 To circumvent this problem, a model has been established whereby instillation of agarose beads coated with Pseudomonas into the lungs of S489X cftr-/- mice has been shown to result in increased inflammation and mortality compared with that observed in wild-type (WT) mice (13, 14). Login to comment
29 To circumvent this problem, a model has been established whereby instillation of agarose beads coated with Pseudomonas into the lungs of S489X cftr-/- mice has been shown to result in increased inflammation and mortality compared with that observed in wild-type (WT) mice (13, 14). Login to comment

PMID: 11978765 [PubMed] Oceandy D et al: "Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities."

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24 CF mice with the null S489X mutation (Cftrm1UNC ) when infected with P. aeruginosa embedded in agar beads produce more proinflammatory cytokines such as tumor necrosis factor a (TNF-a), macrophage inflammatory protein 2 (mip-2) and KC compared with wild-type animals (23). Login to comment

PMID: 11997458 [PubMed] Schroeder TH et al: "CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation."

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20 Noninbred, homozygous ⌬F508 cftr mice (5) and doubly transgenic S489X cftrFABPhuCFTR mice expressing no murine CFTR protein, but expressing wt human CFTR protein in the gastrointestinal tract from the CFTR gene under the control of the mouse fatty acid binding protein promoter (6), were bred in our facility (7). Login to comment
91 There was no translocation of NF-␬B after intratracheal injection of P. aeruginosa into doubly transgenic S489X cftrFABPhuCFTR mice, which lacked expression of murine CFTR in any tissue but expressed human CFTR in the gastrointestinal tract (ref. Login to comment

PMID: 12007216 [PubMed] Bobadilla JL et al: "Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening."

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112 Jewish 1) 405+1G®A (48.0%) 3) W1282X (17.0%) - - 4 23 Kerem et al. [1995] (Tunisia) 2) DF508 (31.0%) 4) 3849+10KbC®T (4.0%) Jewish 1) G85E 4) G542X - - 6 10 Kerem et al. [1995] (Turkey) 2) DF508 5) 3849+10KbC®T 3) W1282X 6) W1089X Jewish (Yemen) None - - 0 5 Kerem et al. [1995] Lebanon 1) DF508 (35.0%) 6) 4096-28G®A (2.5%) - - 9 40 Desgeorges et al. [1997] 2) W1282X (20.0%) 7) 2789+5G®A (2.5%) 3) 4010del4 (10.0%) 8) M952I (2.5%) 4) N1303K (10.0%) 9) E672del (2.5%) 5) S4X (5.0%) Reunion ∆F508 (52.0%) 1717-1G→A (0.7%) 90.4 81.7 9 138 Cartault et al. [1996] Island Y122X (24.0%) G542X (0.7%) 3120+1G→A (8.0%) A309G (0.7%) A455E (2.2%) 2789+5G→A (0.7%) G551D (1.4%) Saudi North: 3) H139L - - North 1 49 families El-Harith et al. [1997]; Arabia 1) 1548delG 4) L1177X Central 3 Kambouris et al. [1997]; Central: 5) DF508 South 4 Banjar et al. [1999] 1)I1234V 6) 3120+1G®A West 9 2)1548delG 7) 425del42 East 6 3)DF508 8) R553X South: 9) N1303K 1) I1234V East: 2) 1548delG 1) 3120+1G®A 3) 711+1G®T 2) H139L 4) 3120+1G®A 3) 1548delG West: 4) DF508 1) I1234V 5) S549R 2) G115X 6) N1303K Tunisia ∆F508 (17.6%) G85E (2.6%) 58.7 34.5 11 78 Messaoud et al. [1996] G542X (8.9%) W1282X (2.6%) 711+1G→T (7.7%) Y122X (1.3%) N1303K (6.4%) T665S (1.3%) 2766del8NT (6.4%) R47W+D1270N (1.3%) R1066C (2.6%) Turkeye ∆F508 (24.5%) 1066L (1.3%) 80.6 65.0 36 1067/670 Yilmaz et al. [1995]; Estivill et al. 1677delTA (4.1%) E822X (1.3%) [1997]; Onay et al. [1998]; 2789+5G→A (3.9%) 2183+5G→A+2184insA (1.3%) Macek et al. [2002] 2181delA (3.8%) D110H (0.8%) R347H (3.6%) P1013L (0.8%) N1303K (2.9%) 3172delAC (0.8%) 621+1G→T (2.6%) 1259insA (0.8%) G542X (2.6%) M1028I (0.8%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS587 E92K (2.6%) 4005+1G→A (0.7%) A96E (2.6%) W1282X (0.7%) M152V (2.6%) I148T (0.6%) 2183AA→G (2.5%) R1162X (0.6%) 296+9A→T (1.6%) D1152H (0.6%) 2043delG (1.4%) W1098X (0.6%) E92X (1.4%) E831X (0.6%) K68N (1.4%) W496X (0.6%) G85E (1.3%) F1052V (0.5%) R1158X (1.3%) L571S (0.5%) United Arab S549R (61.5%) ∆F508 (26.9%) 88.4 78.1 2 86/52 Frossard et al. [1988]; Emirates Frossard et al. [1999] North/Central/South Americas Argentina ∆F508 (58.6%) N1303K (1.8%) 69.1 47.7 5 326/228 CFGAC [1994]; Chertkoff et al. W1282X (3.9%) 1717-1G→A (0.9%) [1997] G542X (3.9%) Brazilf ∆F508 (47.7%) W1282X (1.3%) 66.8 44.6 10 820/500 CFGAC [1994]; Cabello et al. (total) G542X (7.2%) G85E (1.3%) [1999]; Raskin et al. [1999]; R1162X (2.5%) R553X (0.7%) Bernardino et al. [2000] R334W (2.5%) L206W (0.6%) N1303K (2.4%) 2347delG (0.6%) South East: >∆F508, G542X South: >N1303K Brazil ∆F508 (31.7%) N1303K (2.5%) 42.5 18.1 3 120 Parizotto and Bertuzzo [1997] (Sao Paulo) G542X (8.3%) Canada ∆F508 (59.0%) G542X (0.5%) 98.5 97.0 13 381/200 Rozen et al. [1992]; (Lac St. Jean) 621+1G→T (24.3%) N1303K (0.5%) De Braekeleer et al. [1998] A445E (8.2%) Q890X (0.5%) Y1092X (1.2%) S489X (0.5) 711+1G→T (1.0%) R117C (0.5%) I148T (1.0%) R1158 (0.5%) G85E (0.8%) Canada ∆F508 (71.4%) ∆I507 (1.3%) 90.9 82.6 7 77 Rozen et al. [1992] (Quebec City) 711+1G→T (9.1%) Y1092X (1.3%) 621+1G→T (5.2%) N1303K (1.3%) A455E (1.3%) Canada ∆F508 (70.9%) W1282X (0.9%) 82.0 67.2 10 632 Kristidis et al. [1992] (Toronto) G551D (3.1%) R117H (0.9%) G542X (2.2%) 1717-1G→A (0.6%) 621+1G→T (1.3%) R560T (0.6%) N1303K (0.9%) ∆I507 (0.6%) Chile ∆F508 (29.2%) R553X (4.2%) 33.4 11.2 2 72 Rios et al. [1994] Columbia 1) DF508 (35.4%) 3) N1303K (2.1%) - - 4 48 Restrepo et al. [2000] 2) G542X (6.3%) 4) W1282X (2.1%) Ecuador 1) DF508 (25%) - - 1 20 Paz-y-Mino et al. [1999] (Continued) BOBADILLAETAL. Login to comment

PMID: 12578988 [PubMed] Coleman FT et al: "Hypersusceptibility of cystic fibrosis mice to chronic Pseudomonas aeruginosa oropharyngeal colonization and lung infection."

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19 Cftrtm1Unc -TgN(FABPCFTR) [fatty acid-binding protein (FABP)- CFTR] mice have a stop codon in the murine cftr gene (S489X) but also express human CFTR in the gut epithelium due to transgenic introduction of Cftr under the control of the FABP promoter (10). Login to comment

PMID: 14514520 [PubMed] Van Heeckeren AM et al: "Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection."

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222 L722 Stock Cftrtm1Unc -TgN(FABPCFTR)#Jaw mice bear the S489X mutation in Cftr and the human CFTR transgene driven by the fatty acid binding promoter (FABP), which drives CFTR expression primarily in the intestinal tract. Login to comment

PMID: 14715526 [PubMed] Clarke LL et al: "Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice."

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25 The obstructive syndrome and histopathological appearance are accurately reproduced in CF mouse models, in particular, CFTR knockout mice, such as the S489X mutant mouse (42). Login to comment
54 The experiments in this study used gene-targeted weanling mice (4-6 wk old) that were homozygous for the S489X mutation (cftrtm1Unc ) (42) and maintained on a C57BL/6J background (Ͼ6 generations). Login to comment

PMID: 14764916 [PubMed] Bonora M et al: "Ventilatory responses to hypercapnia and hypoxia in conscious cystic fibrosis knockout mice Cftr-/-."

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27 Female and male 129/BC mice heterozygous for the S489X mutation were crossed to obtain homozygous S489X mice (7). Login to comment
33 Mice homozygous for the S489X mutation were classified as Cftr-/- , mice heterozygous for the S489X mutation as Cftrϩ/- , and wild-type mice as Cftrϩ/ϩ . Login to comment
149 However, minor changes in the respiratory tract have been described in the models with the S489X Cftr mutation. Login to comment

PMID: 15007059 [PubMed] Zdebik AA et al: "Additional disruption of the ClC-2 Cl(-) channel does not exacerbate the cystic fibrosis phenotype of cystic fibrosis transmembrane conductance regulator mouse models."

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57 EXPERIMENTAL PROCEDURES Mice-ClC-2 knock-out mice, described previously (24) and backcrossed for seven generations into C57Bl6, were mated with heterozygous CFTR ⌬F508 mice (6) (CFTRkth , called Cftr⌬F/⌬F ) and CFTR S489X mice (5)(CFTRunc , called Cftr-/- ) (26) (both in C57Bl6 background) obtained from Jackson Laboratories. Login to comment

PMID: 15246977 [PubMed] van Heeckeren AM et al: "Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice."

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15 Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and ⌬F508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Login to comment
17 The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-␣, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Login to comment
35 This latter model has shown that the inflammatory response to these P. aeruginosa-laden agarose beads is excessive in cystic fibrosis mice bearing the S489X knockout mutation compared with wild-type littermates (9, 26, 27). Login to comment
49 In these experiments we tested cystic fibrosis mice bearing the ⌬F508, R117H, Y122X, or S489X genotypes, all backcrossed to the common C57BL/6J genetic background, using the mucoid P. aeruginosa agarose bead model to compare their inflammatory responses. Login to comment
51 MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment
57 Cystic fibrosis mice for these strains are indicated by their Cftr mutation (e.g., B6.129P2-Cftrtm1Unc are referred to as cystic fibrosis mice bearing the S489X mutation). Login to comment
61 Cystic fibrosis mice bearing the severe mutations S489X, Y122X, and ⌬F508 were fed the liquid elemental diet Peptamen (Nestle Clinical Nutrition, Deerfield, IL) after weaning, whereas cystic fibrosis mice bearing the mild mutation R117H were fed a standard rodent chow until 1 wk before the start of the experiment at which point they were fed Peptamen until the termination of the experiment. Login to comment
78 At 3 wk of age mice were weighed and then weaned, at which point cystic fibrosis mice bearing the severe Cftr mutations S489X, Y122X, and ⌬F508 were fed a liquid diet. Login to comment
124 Cystic fibrosis mice bearing the severe Cftr mutations Y122X, S489X, and ⌬F508 weighed significantly less (P Ͻ 0.05) than homozygote wild-type controls at 7, 14, and 21 days of life with one exception; cystic fibrosis mice with the Y122X mutation did not differ significantly from wild-type mice at 7 days of age (P Ͼ 0.05), but sample sizes were small. Login to comment
131 The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment
133 Absolute body weight of cystic fibrosis mice before weaning Cftr Genotype Sample Size Age, days 7 14 21 S489X/S489X 14 3.0Ϯ0.6 5.8Ϯ0.9 7.0Ϯ1.1 Y122X/Y122X 4 3.6Ϯ1.7 6.1Ϯ2.3 7.2Ϯ2.5 ⌬F508/⌬F508 10 3.1Ϯ0.9 5.6Ϯ1.2 6.7Ϯ0.8 R117H/R117H 15 4.7Ϯ0.7* 7.1Ϯ0.9* 7.9Ϯ1.8 Data are represented as means Ϯ SD. Login to comment
135 *Significantly different from cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation of the same age as assessed by one-way ANOVA; pairwise comparisons made by Tukey`s test. Login to comment
138 Inflammatory Responses of Cystic Fibrosis Mice Experiments were conducted with each genotype compared in the same experiments to mice bearing the S489X mutation. Login to comment
141 Nevertheless, each genotype can be compared directly with S489X under identical conditions. Login to comment
145 That is, when comparisons were made between mice bearing the S489X mutation and those bearing the Y122X mutation, we combined data from experiments 68, 72, 80, and 94, taking into account any differences between the experiments. Login to comment
146 Similarly, comparisons between mice bearing the S489X mutation and those bearing the ⌬F508 mutation were made by combining data from experiments 68, 75, 90, and 94, and by combining data from experiments 64 and 94, comparisons were made between mice bearing the S489X mutation and those bearing the R117H mutation. Login to comment
149 There were no significant differences in starting weight between cystic fibrosis mice bearing the S489X mutation and those bearing any other Cftr mutation (Fig. 3A). Login to comment
152 After differences between the experiments are taken into consideration (Fig. 3B), weight loss in cystic fibrosis mice bearing the S489X mutation is significantly greater than those bearing the Y122X mutation on days 1, 2, and 3 (P Ͻ 0.05) and significantly less than those bearing the R117H mutation on days 1 and 2 (P Ͻ 0.05). Login to comment
153 There were no significant differences in weight loss between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation. Login to comment
156 When stratifying for experiment, we found significant differences (P Ͻ 0.05) between cystic fibrosis mice bearing the Y122X mutation and those bearing the S489X mutation with Fig. 1. Login to comment
160 Cystic fibrosis mice bearing the S489X mutation were removed from this study to be used in other studies starting at 6 wk of age, and data are censored by death in cystic fibrosis mice bearing the Y122X mutation starting after 6 wk of age. Login to comment
162 L947ROLE regard to TNF-␣ and IL-1beta concentrations in the epithelial lining fluid, although there were no significant differences in cytokine levels between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 or R117H mutations. Login to comment
163 In direct comparisons when stratifying for experiment, we found no significant differences in epithelial lining fluid concentrations of IL-6, MIP-2, KC, LTB4, or PGE2 (P Ͼ 0.05) between mice bearing the S489X mutation and those bearing any of the other Cftr mutations. Login to comment
165 Cystic fibrosis mice bearing the R117H mutation had significantly lower relative neutrophil numbers and greater absolute alveolar macrophage numbers (Table 5) compared with those bearing the S489X mutation in direct comparisons when stratifying for experiment. Login to comment
167 In a comparison of S489X vs. Y122X, ⌬F508, and R117H genotypes, the available sample sizes provide 80% power to detect Fig. 2. Login to comment
171 The difference in nasal PD from the start of the response to 1 min later was determined, and representative tracings are shown to the end of the tracing period from wild type (A), S489X (B), Y122X (C), ⌬F508 (D), and R117H (E) mice. Login to comment
173 Nasal potential differences in wild-type mice and mice bearing mutations in Cftr Cftr Mutation Sample Size Amiloride, mV Chloride-free, Amiloride, Forskolin, mV None 3 -2.1Ϯ1.5 14.4Ϯ1.2 S489X 7 -13.2Ϯ5.1* -2.6Ϯ2.2* Y122X 3 -10.9Ϯ3.7 -0.8Ϯ1.3* ⌬F508 6 -7.6Ϯ3.5 -1.8Ϯ0.8* R117H 3 -8.5Ϯ0.1 0.1Ϯ0.8* Data are means Ϯ SD. Login to comment
176 Starting sample sizes in each experiment Experiment Mouse Strain by Cftr Mutation S489X Y122X ⌬F508 R117H 64 9 (1) 0 0 9 (1) 68 8 4 5 0 72 8 (1)* 8 0 0 75 7 (1) 0 11 (1) [2]* 0 80 9* 10 0 0 90† 10 0 10 0 94† 9 7* 9 (1) 9 (1)* Total 60 (3) 29 35 (2) [2] 18 (2) The number of mice that died due to surgical complications or pulmonary obstruction is noted in parentheses and brackets, respectively. Login to comment
181 When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment
183 Kruskal-Wallis tests were performed only on data from the S489X mice, comparing responses across experiments for the weight, cytokine, and cell count data. Login to comment
194 *Significantly different from cystic fibrosis mice bearing the S489X mutation at the same time point, after differences between experiments are taken into consideration. Login to comment
196 Inflammatory mediators in epithelial lining fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X TNF-␣ 7.8* (5.1/13.1) 10.3 (6.0/14.3) 8.0 (6.7/12.2) 9.5 (7.2/12.4) 14.7 (5.9/23.5) 13.6 (9.1/22.3) IL-1beta 14.5* (5.6/19.3) 16.0 (7.5/25.7) 14.4 (10.0/20.2) 16.0 (8.2/20.2) 12.2 (3.0/32.6) 18.2 (8.1/24.3) IL-6 12.3 (5.9/28.4) 14.6 (7.9/33.1) 11.9 (7.8 (22.8) 13.4 (8.0/19.2) 16.6 (7.3/24.7) 20.8 (9.0/62.4) MIP-2 55.8 (12.1/86.3) 66.9 (34.8/107.3) 90.8 (58.8/129.7) 102.2 (48.0/140.8) 55.6 (21.2/202.5) 96.0 (37.6/131.5) KC 4.1 (3.3/5.5) 5.9 (3.5/7.8) 5.4 (4.1/9.4) 6.3 (4.8/8.6) 12.8 (5.0/15.8) 7.4 (4.6/10.7) LTB4 1.2 (0.2/3.3) 2.3 (1.3/7.8) 5.9 (3.6/10.5) 7.1 (2.3/10.5) 2.1 (1.2/16.7) 2.3 (1.3/7.8) PGE2 4.4 (2.3/6.0) 8.0 (4.4/12.2) 11.4 (7.3/16.6) 12.9 (7.6/22.0) 9.2 (5.9/13.5) 8.0 (4.4/12.2) Data are pooled from available data, are represented as the median (25th/75th percentiles), and are expressed as ng/ml epithelial lining fluid (ELF). Login to comment
198 *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment
203 This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment
207 Correcting the Cftr defect in the gut of cystic fibrosis mice bearing the S489X mutation, by transgenic provision of human CFTR driven by the fatty acid binding protein promoter, results in a much more robust cystic fibrosis mouse that grows normally and does not have intestinal obstruction on a diet of normal mouse chow. Login to comment
210 In this model of lung infection and inflammation, four different genotypes of cystic fibrosis mice were tested: two knockout mice, Y122X and S489X; mice homozygous for the major processing mutation in cystic fibrosis, ⌬F508; and mice homozygous for a channel mutant, R117H, which reaches the plasma membrane but does not function normally. Login to comment
211 None of the cystic fibrosis mice studied here grows as well as their wild-type littermates, although the cystic fibrosis mice bearing the R117H mutation maintain weight better at week 1 of life. Login to comment
213 Previous results indicate that cystic fibrosis mice bearing the ⌬F508 mutation display the cystic fibrosis phenotype established for the S489X knockout mouse (5) with regard to the nasal potential difference (3, 12). Login to comment
214 Here we show that cystic fibrosis mice bearing the Cftr mutations S489X, ⌬F508, Y122X, and R117H on the congenic C57BL/6J background also display the cystic fibrosis electrophysiological phenotype. Login to comment
227 Cell numbers in BAL fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X %AM 5.3 (2.8/41.1) 6.0 (4.3/11.9) 5.8 (3.3/8.7) 4.3 (2.4/5.9) 12.7 (5.8/25.0) 4.7 (4.3/7.4) %PMN 94.3 (58.9/96.9) 94.0 (87.3/95.6) 94.2 (91.3/96.7) 95.3 (94.1/97.6) 87.3* (75.0/94.2) 95.0 (92.6/95.3) %Lymph 0.0 (0.0/0.7) 0.0 (0.0/1.3) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) AM/ml 36 (12/66) 34 (15/72) 47 (23/88) 60 (12/126) 104* (32/168) 78 (56/102) PMN/ml 787 (14/1,449) 801 (422/1,528) 850 (396/1,310) 1,378 (423/1,714) 857 (415/1,549) 1,520 (1,125/1,716) Data are pooled from available data and are represented as the median (25th/75th percentiles). Login to comment
230 *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment
242 Each genotype was studied at the same time as cystic fibrosis mice bearing the S489X mutation, so that direct comparisons are possible (same preparation of agarose beads, same circumstances in the animal facility), and there were no substantive differences between cystic fibrosis mice bearing the S489X mutation and any other genotype. Login to comment
243 Only the cystic fibrosis mice bearing the Y122X mutation differed in two cytokines from S489X, and this may represent the effect of multiple comparisons, rather than a real difference between them. Login to comment
13 Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and ⌬F508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Login to comment
33 This latter model has shown that the inflammatory response to these P. aeruginosa-laden agarose beads is excessive in cystic fibrosis mice bearing the S489X knockout mutation compared with wild-type littermates (9, 26, 27). Login to comment
46 In these experiments we tested cystic fibrosis mice bearing the ⌬F508, R117H, Y122X, or S489X genotypes, all backcrossed to the common C57BL/6J genetic background, using the mucoid P. aeruginosa agarose bead model to compare their inflammatory responses. Login to comment
48 MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment
54 Cystic fibrosis mice for these strains are indicated by their Cftr mutation (e.g., B6.129P2-Cftrtm1Unc are referred to as cystic fibrosis mice bearing the S489X mutation). Login to comment
58 Cystic fibrosis mice bearing the severe mutations S489X, Y122X, and ⌬F508 were fed the liquid elemental diet Peptamen (Nestle Clinical Nutrition, Deerfield, IL) after weaning, whereas cystic fibrosis mice bearing the mild mutation R117H were fed a standard rodent chow until 1 wk before the start of the experiment at which point they were fed Peptamen until the termination of the experiment. Login to comment
75 At 3 wk of age mice were weighed and then weaned, at which point cystic fibrosis mice bearing the severe Cftr mutations S489X, Y122X, and ⌬F508 were fed a liquid diet. Login to comment
121 Cystic fibrosis mice bearing the severe Cftr mutations Y122X, S489X, and ⌬F508 weighed significantly less (P Ͻ 0.05) than homozygote wild-type controls at 7, 14, and 21 days of life with one exception; cystic fibrosis mice with the Y122X mutation did not differ significantly from wild-type mice at 7 days of age (P Ͼ 0.05), but sample sizes were small. Login to comment
128 The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment
130 Absolute body weight of cystic fibrosis mice before weaning Cftr Genotype Sample Size Age, days 7 14 21 S489X/S489X 14 3.0Ϯ0.6 5.8Ϯ0.9 7.0Ϯ1.1 Y122X/Y122X 4 3.6Ϯ1.7 6.1Ϯ2.3 7.2Ϯ2.5 ⌬F508/⌬F508 10 3.1Ϯ0.9 5.6Ϯ1.2 6.7Ϯ0.8 R117H/R117H 15 4.7Ϯ0.7* 7.1Ϯ0.9* 7.9Ϯ1.8 Data are represented as means Ϯ SD. Login to comment
132 *Significantly different from cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation of the same age as assessed by one-way ANOVA; pairwise comparisons made by Tukey`s test. Login to comment
142 That is, when comparisons were made between mice bearing the S489X mutation and those bearing the Y122X mutation, we combined data from experiments 68, 72, 80, and 94, taking into account any differences between the experiments. Login to comment
143 Similarly, comparisons between mice bearing the S489X mutation and those bearing the ⌬F508 mutation were made by combining data from experiments 68, 75, 90, and 94, and by combining data from experiments 64 and 94, comparisons were made between mice bearing the S489X mutation and those bearing the R117H mutation. Login to comment
150 There were no significant differences in weight loss between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation. Login to comment
157 Cystic fibrosis mice bearing the S489X mutation were removed from this study to be used in other studies starting at 6 wk of age, and data are censored by death in cystic fibrosis mice bearing the Y122X mutation starting after 6 wk of age. Login to comment
159 L947ROLE regard to TNF-␣ and IL-1beta concentrations in the epithelial lining fluid, although there were no significant differences in cytokine levels between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 or R117H mutations. Login to comment
164 In a comparison of S489X vs. Y122X, ⌬F508, and R117H genotypes, the available sample sizes provide 80% power to detect Fig. 2. Login to comment
168 The difference in nasal PD from the start of the response to 1 min later was determined, and representative tracings are shown to the end of the tracing period from wild type (A), S489X (B), Y122X (C), ⌬F508 (D), and R117H (E) mice. Login to comment
170 Nasal potential differences in wild-type mice and mice bearing mutations in Cftr Cftr Mutation Sample Size Amiloride, mV Chloride-free, Amiloride, Forskolin, mV None 3 -2.1Ϯ1.5 14.4Ϯ1.2 S489X 7 -13.2Ϯ5.1* -2.6Ϯ2.2* Y122X 3 -10.9Ϯ3.7 -0.8Ϯ1.3* ⌬F508 6 -7.6Ϯ3.5 -1.8Ϯ0.8* R117H 3 -8.5Ϯ0.1 0.1Ϯ0.8* Data are means Ϯ SD. Login to comment
178 When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment
180 Kruskal-Wallis tests were performed only on data from the S489X mice, comparing responses across experiments for the weight, cytokine, and cell count data. Login to comment
191 *Significantly different from cystic fibrosis mice bearing the S489X mutation at the same time point, after differences between experiments are taken into consideration. Login to comment
193 Inflammatory mediators in epithelial lining fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X TNF-␣ 7.8* (5.1/13.1) 10.3 (6.0/14.3) 8.0 (6.7/12.2) 9.5 (7.2/12.4) 14.7 (5.9/23.5) 13.6 (9.1/22.3) IL-1beta 14.5* (5.6/19.3) 16.0 (7.5/25.7) 14.4 (10.0/20.2) 16.0 (8.2/20.2) 12.2 (3.0/32.6) 18.2 (8.1/24.3) IL-6 12.3 (5.9/28.4) 14.6 (7.9/33.1) 11.9 (7.8 (22.8) 13.4 (8.0/19.2) 16.6 (7.3/24.7) 20.8 (9.0/62.4) MIP-2 55.8 (12.1/86.3) 66.9 (34.8/107.3) 90.8 (58.8/129.7) 102.2 (48.0/140.8) 55.6 (21.2/202.5) 96.0 (37.6/131.5) KC 4.1 (3.3/5.5) 5.9 (3.5/7.8) 5.4 (4.1/9.4) 6.3 (4.8/8.6) 12.8 (5.0/15.8) 7.4 (4.6/10.7) LTB4 1.2 (0.2/3.3) 2.3 (1.3/7.8) 5.9 (3.6/10.5) 7.1 (2.3/10.5) 2.1 (1.2/16.7) 2.3 (1.3/7.8) PGE2 4.4 (2.3/6.0) 8.0 (4.4/12.2) 11.4 (7.3/16.6) 12.9 (7.6/22.0) 9.2 (5.9/13.5) 8.0 (4.4/12.2) Data are pooled from available data, are represented as the median (25th/75th percentiles), and are expressed as ng/ml epithelial lining fluid (ELF). Login to comment
195 *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment
200 This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment
204 Correcting the Cftr defect in the gut of cystic fibrosis mice bearing the S489X mutation, by transgenic provision of human CFTR driven by the fatty acid binding protein promoter, results in a much more robust cystic fibrosis mouse that grows normally and does not have intestinal obstruction on a diet of normal mouse chow. Login to comment
224 Cell numbers in BAL fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X %AM 5.3 (2.8/41.1) 6.0 (4.3/11.9) 5.8 (3.3/8.7) 4.3 (2.4/5.9) 12.7 (5.8/25.0) 4.7 (4.3/7.4) %PMN 94.3 (58.9/96.9) 94.0 (87.3/95.6) 94.2 (91.3/96.7) 95.3 (94.1/97.6) 87.3* (75.0/94.2) 95.0 (92.6/95.3) %Lymph 0.0 (0.0/0.7) 0.0 (0.0/1.3) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) AM/ml 36 (12/66) 34 (15/72) 47 (23/88) 60 (12/126) 104* (32/168) 78 (56/102) PMN/ml 787 (14/1,449) 801 (422/1,528) 850 (396/1,310) 1,378 (423/1,714) 857 (415/1,549) 1,520 (1,125/1,716) Data are pooled from available data and are represented as the median (25th/75th percentiles). Login to comment
239 Each genotype was studied at the same time as cystic fibrosis mice bearing the S489X mutation, so that direct comparisons are possible (same preparation of agarose beads, same circumstances in the animal facility), and there were no substantive differences between cystic fibrosis mice bearing the S489X mutation and any other genotype. Login to comment
240 Only the cystic fibrosis mice bearing the Y122X mutation differed in two cytokines from S489X, and this may represent the effect of multiple comparisons, rather than a real difference between them. Login to comment

PMID: 15279681 [PubMed] Cohen JC et al: "The "Goldilocks effect" in cystic fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse."

No. Sentence Comment

22 PV curves were measured in triplicate, starting from positive end-expiratory pressure (PEEP) values of 0, 3, and 6 cmH2O in S489X mice at 30-60 days of age following genotyping for the normal and mutant cftr alleles. Login to comment
79 Methods Mouse strain The S489X mouse 5th generation backcross to C57Bl/6 has been maintained by random mating for the past 8 years. Login to comment
81 Mice 30-60 days of age from our S489X mouse colony were genotyped for the normal and mutant cftr alleles. Login to comment

PMID: 15536480 [PubMed] Modiano G et al: "A large-scale study of the random variability of a coding sequence: a study on the CFTR gene."

No. Sentence Comment

33 In the Tajima`s test,19 the null hypothesis of neutrality is rejected if a statistically significant difference between p Common and rare nonsynonymous and synonymous cSNSs G Modiano et al European Journal of Human Genetics Table 1 List of the 61 cSNSsa encountered in the present survey The random samples of genes (and the technique utilized) cSNS variants found NE Italy (DGGE) Central Italy (DGGE) Southern France (DGGE) Northern France (DHPLC) Spain (SSCA) Czechia (DGGE) Hb Â 104 Exon Exon Length (bp) Ref. no. SNS SASc 1st 100d 2nd 500 1st 100d 2nde 1st 100d 2nd 500 1st 100 2nde 82d 72 Abs. Freq. Total sample size q Â 104 se Â 104 NSf Sf 1g 53 0 0 0 0 0/452 0 924 2 111 1 223C4T R31C 1 1 1/500 1 1 0 0/450 0 5 (11) 1 932 (2 432) 45.23 13.61 90 2 224G4T R31L 0 0 0/500 0 0 0 1/450 0 1 1 932 5.17 5.17 10 3 257C4T S42F 0 0 1/500 0 0 0 0/450 0 1 1 932 5.17 5.17 10 3 109 4 334A4G K68E 1 0 0 0/498 0 0 0 0/452 0 0 1 2 504 3.99 3.99 8 5 352C4T R74W 0 0 0 0/498 0 0 0 1/452 0 0 1 2 504 3.99 3.99 8 6 356G4A R75Q 1 7 1 7/498 2 9 2 9/452 0 2 40 (40) 2 504 (2 544) 157.23 24.66 310 7 386G4A G85E 0 0 1 1/498 0 0 0 0/452 0 0 2 2 504 7.99 5.65 16 4 216 8 482G4A R117H 0 0 0 0/292 0 2 0 1/456 0 0 3 2 302 13.03 7.52 26 9 528T4G I132M 0 0 0 0/292 0 0 0 1/456 0 0 1 2 302 4.34 4.34 8 10 575T4C I148T 1 2 0 1/292 0 0 0 1/456 0 1 6 2 302 26.06 10.63 52 5 90 11 640C4T R170C 0 0 0 0/6 0 0 1/448 0 1 1 436 6.96 6.96 14 12 641G4A R170H 1 1 0 0/6 0 0 2/448 0 4 (4) 1 436 (1 930) 20.73 10.35 41 6a 164 0 0 0/6 0 0 0/432 0 0 992 6b 126 0 0 0/6 0 0 0/454 0 942 7 247 0 0 0/6 0 0 0/796 0 1 284 8 93 13 1281G4A L383 0 0 0 0/6 0 0 1/456 0 0 1 1 516 6.60 6.60 13 9 183 14 1402G4A G424S 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 15 1459G4T D443Y 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 10 192 16 1540A4G M470Vh 42 197 30 37/96 39 199 (i) (i) 27 571(736) 1 484 (1 912) 3849.37 111.28 4 735 17 1598C4A S489X 0 0 0 0/96 0 0 0 1/796 0 1 2 374 4.21 4.21 8 18 1648A4G I506V 1 0 0 0/96 0 0 0 0/796 0 1 2 374 4.21 4.21 8 19 1655T4G F508C 0 1 0 0/96 0 0 0 1/796 0 2 2 038 8.42 5.96 17 20 1716G4A Q528 2 16 1 0/96 0 19 i I 5 43 (58) 1 478 (2 024) 286.56 37.08 557 11 95 21 1756G4T G542X 0 2 0 0/134 0 0 0/796 0 0 2 1 984 10.08 7.12 20 22 1764T4G G544 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 23 1784G4A G551D 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 12 87 24 1816G4A V562I 0 0 0 0 1 0 0/450 0 0 1 (1) 2 004 (2 504) 3.99 3.99 8 25 1816G4C V562L 0 0 0 1 0 0 1/450 0 0 2 (3) 2 004 (2 504) 11.98 6.91 24 26 1859G4C G576A 1 2 0 1 11 0 8/450 0 0 23 (27) 2 004 (2 538) 106.38 20.36 213 13 724j 449 27 1997G4A G622D 0 0 0/80 0/96 1 0 0 0/444 0 1 2 002 5.00 5.00 10 28 2082C4T F650 1 0 0/80 0/20 0 0 0 0/444 0 1 (1) 1 926 (2 412) 4.15 4.15 8 29 2134C4T R668C 1 2 0/80 0/96 1 11 0 12/444 0 27(32) 2 002 (2 558) 125.10 21.98 247 275 30 2377C4T L748 0 0 0/6 0 1 1 388 25.77 25.77 52 14a 129 31 2670G4A W846X 0 0 0/6 0 1 0/452 0/80 0 1 1 010 9.90 9.90 20 32 2694T4G T854 33 23 0/6 33 38 149/452 14/80 11 301 1 010 2980.20 143.92 4 184 33 2695G4A V855I 0 0 0/6 0 0 1/452 0/80 0 1 1 010 9.90 9.90 20 14b 38 0 0 0 0/520 0 0 0 0/446 0 2 448 15 251 34 2816G4C S895T 0 0 0/6 0 0 2/436 0 0 2 996 20.08 14.18 40 35 2831A4C N900T 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 36 2988G4C M952I 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 37 3030G4A T966 (2)k (1)k 0 6/436 0 6 (25)k 618 (1814)k 137.82 27.37 272 38 3032T4C L967S 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 16 80 0 0 0/498 0 0 0/450 0 0 1 502 17a 151 39 3123G4C L997F 0 2 2 1/494 0 7 1 4/454 0 0 17 2 502 67.95 16.42 135 40 3157G4A A1009T 0 2 0 0/494 0 0 0 0/454 0 0 2 2 502 7.99 5.65 16 41 3212T4C I1027T 1 0 0 0/494 0 0 0 0/454 0 0 1 2 502 4.00 4.00 8 17b 228 42 3286T4G F1052V 1 1 0 1/194 0 0 0 0/452 0 0 3 (3) 2 200 (2 240) 13.39 7.73 27 43 3337G4A G1069R 0 1 0 0/194 0 0 0 0/452 0 0 1 2 200 4.55 4.55 9 CommonandrarenonsynonymousandsynonymouscSNSs GModianoetal 186 EuropeanJournalofHumanGenetics 44 3345G4T Q1071H 0 0 0 0/194 0 1 0 0/452 0 0 1 2 200 4.55 4.55 9 45 3417A4T T1995 1 3 0 0/194 1 1 0 0/452 0 0 6 (8) 2 200 (2 506) 31.92 11.27 64 46 3419T4G L1096R 0 0 0 0/194 1 0 0 0/452 0 0 1 2 200 4.55 4.55 9 47 3477C4A T1115 0 0 0 0/194 0 0 0 1/452 0 0 1 2 200 4.55 4.55 9 18 101 48 3523A4G I1131V 0 0 1 0/10 0 0 0/448 0 0 1 (2) 1 512 (1 908) 10.48 7.07 21 49 3586G4C D1152H 0 0 0 0/10 0 0 1/448 0 0 1 1 512 6.61 6.61 13 19 249 50 3617G4T R1162L 0 0 1 1/494 0 0/260 0 0/454 0 0 2 2 262 8.84 6.25 18 51 3690A4G Q1186 0 0 0 0/494 0 0/260 0 0/454 1 0 1 2 262 4.42 4.42 9 52 3813A4G L1227 0 1 0 0/494 0 0/260 0 0/454 0 0 1 2 262 4.42 4.42 9 53 3837T4G S1235R 1 1 0 1/494 0 4/260 0 7/454 0 1 15 (15) 2 262 (2 310) 69.94 16.71 140 20 156 54 4002A4G P1290 2 3 0/6 3 5 18/454 3/80 2 36 1 012 357.73 58.22 690 21 90 55 4009G4A V1293I 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 56 4029A4G T1299 1 0 0/6 0 1/300 0 1/456 0 0 3 (8) 1 316 (2 330) 34.33 12.12 69 57 4041C4G N1303K 1 0 0/6 0 0/300 0 0/456 0 0 1 1 316 7.60 7.60 15 58 4085T4C V1318A 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 22 173 0 0 0/18 0 0 0/450 0 0 1 022 23 106 0 0 0 0/6 0 0 0/448 0 1 436 24l 198+3 59 4404C4T Y1424 1 0 0/6 1 2 5/420 0 2 11 (32) 980 (2 516) 127.19 22.34 251 60m 4521G4A Q1463 (21) (16) (3/32) (14/80) (30) (94/420) 15/76 (17) 15 (227) 76 (1052) 2142.86 131.07 3 367 61 4563T4C D1477 0 0 0/6 0 1 0/420 0 0 1 980 10.20 10.20 20 Totals 6 525 9 584 16 109 The bracketed figures include also the RFLP analysis data (see Materials and methods); the NE Italy, Central Italy, Southern and Northern France are each subdivided into two samples where the 1st is made up of 100 genes. 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PMID: 15650130 [PubMed] Simpson JE et al: "Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum."

No. Sentence Comment

50 The experiments in this study used mice 8-16 wks old that were either homozygous for the S489X mutation of the murine homolog of CFTR (cftrtm1UNC ) or homozygous for the mCFTR ⌬F508 mutation (cftrtm1Kth ) and maintained on a C57BL/6J background (54, 64). Login to comment
51 The experiments in this study used mice 8-16 wks old that were either homozygous for the S489X mutation of the murine homolog of CFTR (cftrtm1UNC ) or homozygous for the mCFTR ⌬F508 mutation (cftrtm1Kth ) and maintained on a C57BL/6J background (54, 64). Login to comment

PMID: 16078047 [PubMed] van de Vosse E et al: "Susceptibility to typhoid fever is associated with a polymorphism in the cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

12 A CFTR null allele (S489X) in mice was found to confer resistance to cholera toxin (Gabriel et al. 1994), however, this particular mutation is very rare in humans (found in one patient) and may not be comparable to F508del, W1282X and 1677del TA in survival in a population. Login to comment

PMID: 16614351 [PubMed] Pan J et al: "Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice."

No. Sentence Comment

131 Because NEB represent airway O2 sensors (analogous to carotid body chemoreceptors) involved in the control of breathing (2, 4), the previously mentioned abnormalities could partly account for recent observation of decreased ventilatory responses to hypoxia observed in Cftr-/- mice using whole-body plethysmography (20) and decreased compliance and increased airway resistance in the S489X Cftr-/- mouse (21). Login to comment

PMID: 16675347 [PubMed] Saadane A et al: "Acute Pseudomonas challenge in cystic fibrosis mice causes prolonged nuclear factor-kappa B activation, cytokine secretion, and persistent lung inflammation."

No. Sentence Comment

28 METHODS Mice Male CF-KO mice 7 to 10 weeks old homozygous for the S489X (B6.129P2-CftrtmtUnc ) (null) mutation17 and normal wt/wt (WT) littermates were used after genotyping by polymerase chain reaction on tail snip DNA. Pups were weaned at 10 days of life and fed Peptamen (Clintech Nutrition Co, Deerfield, Ill) to avoid intestinal obstruction.18 All mice were maintained in sterile microisolator cages with sterile water. Login to comment

PMID: 16740913 [PubMed] Jin R et al: "The cystic fibrosis transmembrane conductance regulator (Cftr) modulates the timing of puberty in mice."

No. Sentence Comment

131 Methods: To examine this hypothesis, we investigated pubertal timing (as assessed by vaginal opening (VO)) in a mouse model of CF (Cftrtm1Unc ) engineered to produce a truncated Cftr mRNA and referred to as S489X. Login to comment
146 First, B6 mice heterozygous for the S489X mutation that generates a stop codon in the coding sequence of exon 10 of Cftr (B6.129P2-Cftrtm1Unc /J),20 were purchased from Jackson Laboratory (Bar Harbor, ME; stock no. Login to comment
148 This mutation leads to a truncated protein product similar to many human CF mutations.20 Heterozygous S489X mice (referred to as S489X2 /S489X+ ) were bred and the progeny homozygote, heterozygote, or negative (wild type, WT) for the S489X mutation were studied. Login to comment
153 In the colony described here, the DF508 and S489X lines are backcrossed to B6 mice every five generations to ensure genetic fidelity and prevent genetic drift. Login to comment
154 Live animals were genotyped at 7 days after date of birth by PCR analysis of DNA extracted from a toe removed for mouse identification.24 WT mice from the S489X or DF508 matings are both predicted to be indistinguishable from B6 mice due to the backcrossing of these alleles. Login to comment
155 Because there were no observable phenotypic differences between the WT mice generated from the two mating strategies (mean timing of vaginal opening (VO) 31.3¡1.4 days for S489X WT (n = 14) and 31.3¡1.5 days for DF508 WT (n = 12)), WT animals were assessed as a single group. Login to comment
157 For the S489X mice, the p values for comparison of homozygote and heterozygote mice to WT mice were ,0.00001 and 0.00004, respectively; for the DF508 mice, the p values for comparison of homozygote and heterozygote mice to WT mice were 0.00007 and 0.0002, respectively. Login to comment
172 S489X- /S489X+ 100 90 70 80 60 40 50 30 20 0 10 80 Age (days) S489X- /S489X - MicewithVO(%) 20 7570656055504540353025 WT Figure 1 Comparison of the timing of vaginal opening (VO) for S489X mice. Login to comment
180 RESULTS VO occurred significantly later in S489X homozygous knockout (S489X2 /S489X2 ) mice than in WT B6 mice (table 1, fig 1). Login to comment
181 The S489X CF mice also grew more slowly than WT mice and had not even reached a mean weight of 15 g by 50 days of age; thus, despite the delay in VO, the S489X2 / S489X2 mice experienced VO at significantly lower mean body weights than the WT animals. Login to comment
182 To investigate the direct effects of Cftr on pubertal timing without the confounding effects of chronic disease, mice heterozygous for the S489X mutation were examined. Login to comment

PMID: 16763223 [PubMed] Velsor LW et al: "Mitochondrial oxidative stress in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice."

No. Sentence Comment

40 Animal Care and Use This study compared the mitochondrial consequences of the CFTR mutation from two congenic CFTR KO strains (S489X and FABP breeding stock were kind gifts from Dr. Anna van Heeckeren, Case Western Reserve University, Cleveland, OH) with C57B6 control mice. Login to comment
41 In the S489X congenic C57B6 strain, a mutation creates a stop codon and produces a truncated CFTR protein (19). Login to comment
43 To avoid this problem, S489X mice are maintained on a liquid diet (Peptamen; Nestle, Glendale, CA). Login to comment
45 With the exception of the liquid diet for the S489X mice, all mice were provided solid mouse chow and autoclaved tap water ad libitum. Login to comment
138 In the S489X CFTR KO mice on a liquid diet, mitochondrial GSH concentrations were decreased 43% compared with controls. Login to comment
148 Mitochondria isolated from the lungs of S489X and FABP CFTR KO mice displayed significantly lower levels of GSH than mitochondria from control mice. Login to comment
206 Both S489X and FABP CFTR KO mice had significantly lower mitochondrial GSH concentrations than control mice. Login to comment
208 Between the two CFTR KO mouse strains, however, the FABP GSH levels were considerably lower than the levels in the S489X mice. Login to comment
209 The S489X mutation generates a stop codon and produces a truncated protein (19). Login to comment
210 The S489X CFTR KO mouse must be maintained on a liquid diet to prevent fatal bowel obstructions. Login to comment

PMID: 16804061 [PubMed] Stalvey MS et al: "Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury."

No. Sentence Comment

38 RESEARCH DESIGN AND METHODS We used mice with the CFTR S489X-/- neo insertion, developed initially at the University of North Carolina (20). Login to comment
42 CFTR S489X-/- FABP-hCFTRϩ/ϩ (hereafter designated CFTR-/- ), C57BL/6J, and FVB/NJ mice were housed under specific pathogen-free conditions at the University of Florida Animal Care Services according to National Institutes of Health guidelines and allowed food and water ad libitum. Login to comment
56 CFTR S489X-/- animals (4-6 weeks old) were sensitized to Aspergillus fumigatus crude protein extract (Greer Laboratories, Lenoir, NC). Login to comment
66 Lung, intestine, and pancreatic mRNA from CFTR S489X-/- FABP-hCFTR؉/؉ mice was used to analyze the expression pattern of the hCFTR driven by the FABP promoter by RT-PCR, using gene-specific primers for hCFTR and B-actin control primers. Login to comment
180 Interestingly, the CFTR S489X-/- FABP-hCFTRϩ/ϩ model used in this experiment has not been previously reported to have pancreatic disease. Login to comment

PMID: 16906518 [PubMed] Cohen JC et al: "Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis."

No. Sentence Comment

263 CFTR knockouts, S489X mice 5th generation backcross, with C57Bl/6 were used. Login to comment

PMID: 17108316 [PubMed] Magenheimer BS et al: "Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation."

No. Sentence Comment

35 Cftrm1UNC (S489X) mice produce no stable CFTR mRNA or protein and therefore are considered to have a null phenotype (32,33). Login to comment

PMID: 17272824 [PubMed] Saadane A et al: "Parthenolide inhibits IkappaB kinase, NF-kappaB activation, and inflammatory response in cystic fibrosis cells and mice."

No. Sentence Comment

61 These are stock CftrtmtUnc -TgN(FABPCFTR)#Jaw mice bearing the S489X mutation in cftr and also a transgene for full-length human CFTR driven by the fatty acid-binding promoter (FABP), which restores CFTR function in the intestinal tract. Login to comment

PMID: 17369290 [PubMed] Kariya C et al: "A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration."

No. Sentence Comment

43 C57BL/6J congenic Cftr KO (S489X) mice (22.3 Ϯ 0.2 g) that possessed the S489X mutations in the murine equivalent to CFTR (47) and C57BL/6J congenic gut-corrected Cftr KO-Tg mice (23.7 Ϯ 0.3 g) that had intestinal specific expression of normal human Cftr driven by the fatty acid binding promoter (57) were employed in these studies. Login to comment

PMID: 17805210 [PubMed] Cottart CH et al: "Impact of nutrition on phenotype in CFTR-deficient mice."

No. Sentence Comment

21 Animals used in this study were male CF mice homozygous for the S489X mutation (Cftrtm1Unc ) (6) back-crossed into C57BL/6 (three generations) (Cftr-/- ) and their normal littermates (Cftrϩ/ϩ ). Login to comment

PMID: 18023072 [PubMed] Mueller C et al: "Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene."

No. Sentence Comment

35 Mouse strains The primary CFTR knockout strain used for these studies was the CFTR S489X -/- neo insertion in C57BL/6 mice developed initially at the University of North Carolina [41] and then modified with the transgenic overexpression of gut-specific expression of human CFTR from the fatty acid binding protein (FABP) promoter in order to prevent intestinal obstruction and improve viability [42]. Login to comment
46 Aspergillus sensitization and challenge Five- to six-week old CFTR S489X -/-; FABP-hCFTR (+/+), and C57BL/6J mice were house in the SPF mouse colony of the University of Florida according to NIH guidelines and were allowed food and water ad libitum. Login to comment

PMID: 18325992 [PubMed] Hodges CA et al: "Infertility in females with cystic fibrosis is multifactorial: evidence from mouse models."

No. Sentence Comment

36 Materials and Methods Mouse strains The CF mouse models (Cftr-/- ) used in these studies contained either the Cftrtm1Unc mutation (referred to as the S489X mutation in this manuscript), which generates a stop codon in the coding region of exon 10 of Cftr (32) (Jackson Laboratory, Bar Harbor, ME; stock no. Login to comment
76 Results Fertility in Cftr mutant females To determine the effect of the absence of Cftr on female fertility, two independently derived mouse models of CF (S489X and ⌬F508) and wild-type females were mated to wild-type males for 5 months and examined daily for subsequent birth of litters and number of pups. Login to comment
95 Fertility of wild-type and Cftr mutant females Mouse type Mice, n Females with no litters, n (%) Average litter per month Average no. of pups/litter Wild type 18 0 (0) 0.92 Ϯ 0.13 6.56 Ϯ 2.36 ⌬F508 14 5 (35.7) 0.32 Ϯ 0.26a 3.81 Ϯ 1.43a S489X 10 2 (20) 0.36 Ϯ 0.36a 3.55 Ϯ 1.92a Both mutations 24 7 (29.2) 0.34 Ϯ 0.30a 3.70 Ϯ 1.61a a P Ͻ 0.001 vs. wild type. TABLE 2. Login to comment
96 Uterine and ovarian size in wild-type and Cftr mutant females Mouse type Mice, n Average ovarian weight (mg) Average uterine weight (mg) Average mouse weight (g) 6-8 wk Wild type 7 3.1 Ϯ 0.6 61.4 Ϯ 9.8 19.0 Ϯ 1.45 ⌬F508 6 1.4 Ϯ 0.2a 27.4 Ϯ 6.4a 16.4 Ϯ 1.59b S489X 6 1.8 Ϯ 0.6a 26.6 Ϯ 14.3a 15.9 Ϯ 1.46a Both mutations 12 1.6 Ϯ 0.5a 27.0 Ϯ 10.6a 16.1 Ϯ 1.48a 14-16 wk Wild type 11 4.2 Ϯ 1.1 74.8 Ϯ 11.8 21.6 Ϯ 1.92 ⌬F508 6 2.6 Ϯ 0.6a 45.8 Ϯ 20.5b 18.6 Ϯ 0.88a S489X 8 2.8 Ϯ 0.6a 50.6 Ϯ 17.1b 19.4 Ϯ 1.33a Both mutations 14 2.7 Ϯ 0.6a 48.2 Ϯ 18.2b 19.0 Ϯ 1.11a a P Ͻ 0.005 vs. wild type. Login to comment
123 Wild-type and Cftr mutant females` response to exogenous hormones Mouse type Mice, na Average ovarian weight (mg) Average uterine weight (mg) Average mouse weight (g) Oocytes ovulated, n Wild type 10 7.3 Ϯ 2.3 110 Ϯ 10 21.5 Ϯ 3.08 24.3 Ϯ 9.62 ⌬F508 9 7.5 Ϯ 1.9 110 Ϯ 30 19.8 Ϯ 2.10b 24.2 Ϯ 7.87 S489X 6 6.0 Ϯ 1.7 92 Ϯ 14 19.4 Ϯ 1.85b 18.6 Ϯ 5.64 Both mutations 15 6.9 Ϯ 1.8 100 Ϯ 25 19.6 Ϯ 1.98b 22.2 Ϯ 7.47 a Ovulated oocytes were present in all injected females. Login to comment
125 Characterization of estrous cycle in wild-type and Cftr mutant females Mouse type Mice, n Mice with no estrus, n (%) Total no. of estrus Avg no. of cycles/mousea Cycle lengtha Wild type 14 0 (0) 58 4.14 Ϯ 0.86 5.11 Ϯ 1.58 ⌬F508 18 4 (22.2) 29 2.07 Ϯ 1.20b 11.79 Ϯ 5.64b S489X 12 5 (41.7) 16 2.29 Ϯ 1.44b 11.19 Ϯ 6.29c Both mutations 30 9 (30.0) 45 2.14 Ϯ 1.28b 11.59 Ϯ 5.71b a Calculated using only females with at least one estrous cycle. Login to comment
156 Fertilization of oocytes 48 h after hCG in wild-type and mutant females Mouse type (no. of mice) Total cells, n Cells at stage, % Oocyte One cell Two cell Three cell Four cell Wild type (4) 71 8.5 1.4 76.0 5.6 8.5 ⌬F508 (4) 63 98.4 0 1.6 0 0 S489X (4) 65 100 0 0 0 0 Both mutations 128 99.2 0 0.8 0 0 TABLE 6. Login to comment
157 In vitro fertilization of oocytes from wild-type and Cftr mutant females Mouse type (no. of mice) Fertility rate (%)a Wild type (4) 49 of 72 (68.1) ⌬F508 (4) 52 of 74 (70.3) S489X (4) 46 of 68 (67.6) Both mutations (8) 98 of 142 (69.0) a Fertility rate is expressed as the number two-cell embryos over the total number of oocytes (percent). Login to comment
190 Number of oviductal sperm and capacitation in wild-type and Cftr mutant females Mouse type (no. of mice) Total no. of sperm (average per oviduct) Capacitation (n ϭ 200 per mouse) (%) Wild type (3) 1536 Ϯ 291 100 ⌬F508 (3) 142 Ϯ 105a 100 S489X (3) 132 Ϯ 133a 100 Both mutations (6) 137 Ϯ 114a 100 a P Ͻ 0.001 vs. wild type. Login to comment

PMID: 18344710 [PubMed] Madore AM et al: "Distribution of CFTR mutations in Saguenay- Lac-Saint-Jean: proposal of a panel of mutations for population screening."

No. Sentence Comment

48 Altogether, the six mutations represent 95.89% of the CFTR allele of CF patients in the SLSJ population, whereas the proportions are 86.85, 85.27, and Table 2 Cystic fibrosis mutations present in the four populations studied Mutationa Allelic frequency (number of alleles [%]) Populationb 1 2 3 4 „F508 106 (62.35) 55 (72.37) 398 (72.36) 67 (57.78) 621 ؉ 1G>T 42 (24.71) 6 (7.89) 30 (5.45) 1 (0.85) A455E 12 (7.06) 2 (2.63) 14 (2.55) 1 (0.85) 3199del6 1 (0.59) 1 (1.32) 7 (1.27) 1 (0.85) 711 ؉ 1G>T 1 (0.59) 1 (1.32) 15 (2.73) 1 (0.85) Y1092X 1 (0.59) 1 (1.32) 5 (0.91) 0 R117C 2 (1.18) 0 0 0 ‚I507 1 (0.59) 2 (2.63) 10 (1.82) 0 L206W 1 (0.59) 1 (1.32) 9 (1.64) 0 R1158X 1 (0.59) 0 0 0 S489X 1 (0.59) 0 1 (0.18) 0 R553X 0 2 (2.63) 2 (0.36) 0 R334W 0 1 (1.32) 2 (0.36) 0 G542X 0 0 10 (1.82) 0 G85E 0 0 6 (1.09) 5 (4.24) N1303K 0 0 5 (0.91) 1 (0.85) IVS8-5T 0 0 4 (0.73) 0 W1282X 0 0 3 (0.55) 7 (5.93) R347P 0 0 1 (0.18) 2 (1.69) V520F 0 0 1 (0.18) 0 I1027T 0 0 1 (0.18) 0 R1066C/IVS 0 0 1 (0.18) 0 Q1313X 0 0 1 (0.18) 0 1898ϩ3GϾA 0 0 1 (0.18) 0 2183AAϾG 0 0 1 (0.18) 0 2951insA 0 0 1 (0.18) 0 G551D 0 0 0 2 (1.69) 1525-iG-A 0 0 0 2 (1.69) Y109C 0 0 0 1 (0.85) S549N 0 0 0 1 (0.85) 3154del1G 0 0 0 1 (0.85) UNKNOWN 1 (0.59) 4 (5.26) 20 (3.82) 25 (21.19) Number of alleles genotypedc 170 (100) 76 (100) 550 (100) 118 (100) a The six mutations included in the panels proposed are in bold. Login to comment

PMID: 18388934 [PubMed] Davies LA et al: "Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice."

No. Sentence Comment

11 After vector delivery at 16 days of gestation (equivalent to a 15-to 20-week gestation period in humans), transgene expression is observed in the lung, stomach and gut and is highly correlated with increased fetal breathing movements.4 Several CF transgenic mouse strains have been generated in an attempt to model and evaluate potential treatment strategies, but although these mouse models display many of the -CF-related ion-transport bioelectric defects found in human CF tissues, lung pathology is not recapitulated.5 However, some abnormalities of the CF gastrointestinal tract such as intestinal obstruction are observed in mouse models, where they can be very severe, leading to poor survival.6 The CF knockout mouse strain S489X carrying the Cftrtm1Unc mutation develops intestinal obstruction leading to intestinal perforation, peritonitis and death, resulting in only 5% survival to adulthood,7 although these mice can survive given the appropriate dietary intervention.8 Survival of these mice was also reported to increase dramatically when recombinant adenovirus expressing human CF transmembrane conductance regulator (hCFTR) was delivered via intra-amniotic injection to fetal mice in utero.9 The improved survival was observed even though the adenovirus-mediated CFTR expression was transient, implying that continuous expression of CFTR was not required, therefore challenging the widely held view that it is the absence of the CFTR chloride channel that leads to CF disease in the adult. Login to comment
52 Effect of hCFTR expression on survival of cftr -/- mice Any conclusion regarding the effectiveness of in utero hCFTR expression in correcting the lethal intestinal defect in S489X Cftrtm1Unc /Cftrtm1Unc mice will be wholly dependent on the number of Cftr -/- mice surviving after injection of AdCFTR compared with injection of control AdLacZ vector. Login to comment
57 Finally, to assess the impact of in utero delivery of AdCFTR on the survival of Cftr -/- mice, S489X litters were injected at E16 a pCIKCFTR 195 kd 112 kd Band C Band B AdGFP AdCFTR b 0 -0.1 0.0 Rateof 125 |efflux/min 0.1 0.2 0.3 0.4 0.5 1 2 Time (min) 3 4 Untreated AdLacZ AdCFTR T84 cells c 0 0.01 Untreated AdCFTR 0.1 1 10 100 1,000 hCFTRmRNAcopies/ngRNA (3) (6) (7) (4) (13) (4) (+/+) (+/-) (-/-) d Untreated AdCFTR 0 0.01 0.1 1 10 100 hCFTRmRNAcopies/ngRNA (3) (6) (7) (5) (15) (5) (+/+) (+/-) (-/-) Figure 3 Human cystic fibrosis transmembrane conductance regulator (hCFTR) can be detected in lungs and intestines of S489X Cftrtm1Unc mice following intra-amniotic injection of AdCFTR. Login to comment
63 Expression of hCFTR mRNA was confirmed in vivo following intra-amniotic injection of 108 plaque forming units (pfu)/fetus of AdCFTR at E16 to litters resulting from heterozygote matings of S489X Cftrtm1Unc mice. Login to comment
68 Table 1 In utero injection and survival of S489X Cftrtm1Unc /Cftrtm1Unc mice Untreated AdLacZ 107 pfu AdCFTR 107 pfu AdCFTR 108 pfu Litters 21 17 15 13 Injected 0 142 114 102 Lost NA 11 14 6 Born 156 131 100 96 Day 0 -/- 25 20 20 22 +/- 87 54 45 48 +/+ 42 48 30 25 Unknown 2 9 5 1 Day 100 -/- 4 2 2 3 +/- 70 28 39 29 +/+ 33 28 26 13 Unknown 0 0 0 0 Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; pfu, plaque forming units; NA, not applicable. Login to comment
69 Litters from heterozygote S489X matings were injected with AdLacZ or AdCFTR at 16 days of gestation. Login to comment
97 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival (+/+) (+/-) (-/-) a 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival c 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival Untreated AdLacZ 107 AdCFTR 107 AdCFTR 108 b 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival d Figure 4 Survival of S489X Cftrtm1Unc mice following expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) from AdCFTR. Login to comment
98 Kaplan-Meier plots of S489X Cftrtm1Unc survival following intra-amniotic injections. Login to comment
105 It was unfortunate that the S489X Cftrtm1Unc mouse strain dem- onstratedsuchhighlevelsofmaternalcannibalismandneglectlead- ing to a high mortality of newborn pups of all genotypes, especially following surgical interventions. Login to comment
112 An important difference is the ~20% survival of the Cftr -/- mice observed in our facility compared with a previously reported 5%.14 The intestinal pathology and mortality in CF transgenic mouse models can vary dependent on the precise Cftr knockout mutation, environmental influences and the independent segregation of modifier genes.6,17 The survival chances of one CF mouse model Cftrtm1G551D /Cftrtm1G551D were shown to vary between 27 and 67% depending on undetermined environmental factors in the animal breeding facility.18 Interestingly, when we transferred a subset of our S489X Cftr -/- mice to an alternative animal facility they died of intestinal obstruction within a few days (data not shown). Login to comment
115 Unavoidably, our studies used S489X Cftrtm1Unc CF knockout mice as a 10th generation backcross onto to C57BL/6 background, as opposed to a 5th generation backcross.10 In the accompanying paper, Buckley et al.16 attempted independent verification of the original study, using the same adenoviral vector, but using an alternative knockout CF mouse allele (Cftrtm1Cam /Cftrtm1Cam ) on an inbred genetic background (129 S6Sv/Ev) to minimize genetic heterogeneity and the effects of potential modifier genes. Login to comment
124 The Cftr-mutant UNC mouse strain, S489X (Cftrtm1UNC / Cftrtm1UNC ) was obtained as a 10th generation backcross on a C57BL/6 background (Dr Julia Dorin, MRC Human Genetics Unit, Edinburgh, UK) and was maintained via heterozygote matings in open cages with hardwood sawdust and R&M3 breeders` diet (Special Diet Services, Witham, UK) with weaning at 21 days. Login to comment
142 To generate time-mated pregnancies, heterozygote S489X Cftrtm1Unc females were introduced to cages containing heterozygote males overnight with the following day regarded as day 1 of gestation (E1) in resulting pregnancies. Login to comment

PMID: 18450781 [PubMed] Bonvin E et al: "Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice."

No. Sentence Comment

16 Although CFTR-deficient mice do not develop classical lung disease, some alterations in pulmonary function have been reported in Cftrtm1Unc knockout mice homozygous for the S489X mutation (Cftr-/- ) (Kent et al. 1996; Cohen et al. 2004). Login to comment
32 Animals The study was carried out on two different CF mouse models: (1) Cftrtm1Unc knockout mice homozygous for the S489X-CFTR mutation (Snouwaert et al. 1992), back-crossed into C57BL/6 (three generations) (Cftr-/- ) and their normal littermates (Cftr+/+ ). Login to comment
42 We also performed experiments on newborn homozygous mice for both mutations (S489X and F508del) and their control littermates at 1 day postnatal. Login to comment
144 The high rate of tracheal abnormalities in adult Cftr-/- mice homozygous for the S489X mutation of the CFTR protein, in contrast to that observed in control Cftr+/+ mice, suggests that these abnormalities could be directly Table 2. Login to comment

PMID: 18782827 [PubMed] Kukavica-Ibrulj I et al: "Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies."

No. Sentence Comment

170 Table 2 Cystic fibrosis (CF) mouse models CF mice Mutation/molecular strategy Phenotype/limitation CFTR KO CFTRtm1Unc (Snouwaert et al. 1992) Exon 10 replacement, null mutation, inframe stop Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hgu (Dorin et al. 1992) Exon 10 insertional mutagenesis Intestinal blockage; minor pathology in lungs of one mouse CFTRtm1Cam (Ratcliff et al. 1993) Exon 10 replacement, null mutation Severe intestinal phenotype and high mortality; pathology in lacrimal gland and pancreas of some mice; no lung disease CFTRtm1Bay (O`Neal et al. 1993) Exon 3 insertional duplication, null mutation Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hsc (Rozmahel et al. 1996) Exon 1 replacement, null mutation Severe intestinal phenotype and high mortality; no lung disease Other mutations CFTRtm1Kth (Zeiher et al. 1995) DF508 by exon 10 replacement High mortality and reduction in size, variable pathology of the gastrointestinal tract, normal lung, pancreas, gallbladder, male reproductive tract, lacrimal gland and submandibular glands CFTRtm1Eur (van Doorninck et al. 1995, French et al. 1996) DF508 by exon 10 'hit and run` Normal survival, growth retarded but no abnormalities or stasis of inspissated mucus in lungs, pancreas, liver bile ducts, vas deferens and salivary glands CFTRtm1G551D (Delaney et al. 1996) G551D by exon 11 replacement Moderate phenotype with reduced incidence of intestinal blockage and 67% survival; no lung disease CFTRtm2Hgu (Dickinson et al. 2000) G480C by exon 10 'hit and run` Normal survival, no reduction in body weight, preserved cAMP-mediated Cl2 response, decreased Ca2þ -related Cl2 response CFTR2/2hCFTR-G542X (Du et al. 2002) CFTR2/2 null mutation that also express a human CFTR-G542X stop mutation under control of the intestinal FABP promoter Suppression of the hCFTR-G542X mutation in vivo by aminoglycosides CFTRtm1Unc -TgN(FABPCFTR) (Zhou et al. 1994) Stop codon in the murine CFTR gene (S489X) but also express human CFTR in the gut epithelium (transgenic introduction of CFTR under FABP promoter) Functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion, improved survival Congenic C57BL/6J CFTR2/2 (Durie et al. 2004) Long-lived congenic C57BL/6J CFTR2/2 All organs pathologically affected by the human form of CF Scnn1a-, Scnn1b- and Scnn1c-transgenic mice (Mall et al. 2004) Transgenic mice overexpressing airway-specific ENaC to increase Naþ absorption CF-like lung disease FABP: fatty acid-binding protein, ENaC: epithelial Naþ channels. Login to comment

PMID: 18790990 [PubMed] Manson ME et al: "cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells."

No. Sentence Comment

124 To verify results in an in vivo model, we excised nasal epithelium from S489X Cftr-/- mice and assayed for pCREB content compared with nasal epithelium from age-matched wild-type mice. Login to comment
121 To verify results in an in vivo model, we excised nasal epithelium from S489X Cftr-/- mice and assayed for pCREB content compared with nasal epithelium from age-matched wild-type mice. Login to comment

PMID: 19168701 [PubMed] Yu H et al: "Defective acid sphingomyelinase pathway with Pseudomonas aeruginosa infection in cystic fibrosis."

No. Sentence Comment

79 Animal Experiments The CFTR KO mice are Cftrtm1Unc-TgN(FABPCFTR) (fatty acid-binding protein [FABP]-CFTR) mice that have a stop codon in the murine CFTR gene (S489X) but also express human CFTR in the gut epithelium due to transgenic introduction of CFTR under the control of the FABP promoter (30). Login to comment

PMID: 19475687 [PubMed] Halilbasic E et al: "Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice."

No. Sentence Comment

27 Congenic B6.129P2-Cftrtm1Unc mice, which possess the S489X mutation that blocks transcription of Cftr,13 were fed with Peptamen (Nestle Clinical Nutrition, Deerfield, IL) or with Peptamen containing norUDCA (in a dosage equivalent to that used in Mdr2-/- mice) for 1 week. Login to comment

PMID: 19562038 [PubMed] Trudel S et al: "Peroxiredoxin 6 fails to limit phospholipid peroxidation in lung from Cftr-knockout mice subjected to oxidative challenge."

No. Sentence Comment

46 Animals used in this study were Cftr-knockout (Cftr 2/2) mice homozygous for the S489X mutation (B6;129-Cftrtm1Unc) [16] backcrossed into C57BL/6 (three generations) and their wild-type (Cftr+/+) littermates (CDTA, Orle´ans, France). Login to comment

PMID: 19700644 [PubMed] Nichols DP et al: "The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease."

No. Sentence Comment

122 Electrophysiological phenotype (i.e., nasal potential difference) and inflammatory responses in the lung to P. aeruginosa are very similar in R117H mice and multiple other CF transgenic mice, including mice bearing the ⌬F508 or S489X mutation (40). Login to comment

PMID: 19935781 [PubMed] Mueller C et al: "Modulation of exaggerated-IgE allergic responses by gene transfer-mediated antagonism of IL-13 and IL-17e."

No. Sentence Comment

189 The primary Cftr knockout strain used for these studies was the CFTR S489X-/- neo insertion in C57BL/6 mice developed initially at the University of North Carolina24 and then modified with the transgenic overexpression of gut-specific expression of human CFTR from the fatty acid-binding protein promoter in order to prevent intestinal obstruction and improve viability.25 These mice have then been backcrossed 10 generations onto a C57BL/6 mouse. Login to comment
194 Six to eight-week-old Cftr S489X-/-; fatty acid-binding protein human CFTR+/+, and wild-type lit- termatemicewerehousedinthespecificpathogen-freemousecolonyofthe University of Massachusetts Medical School (Worcester, MA) according to National Institutes of Health guidelines and were allowed food and water ad libitum. Login to comment

PMID: 20154695 [PubMed] Harmon GS et al: "Pharmacological correction of a defect in PPAR-gamma signaling ameliorates disease severity in Cftr-deficient mice."

No. Sentence Comment

154 Eckman, E.A., Cotton, C.U., Kube, D.M. & Davis, P.B. Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment
293 We inbred mice heterozygous for the S489X (B6.129P2-CFTRtm1Unc, or Cftr+/-) mutation for more than ten generations. Login to comment

PMID: 20309575 [PubMed] Gouyer V et al: "The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice."

No. Sentence Comment

37 Cftr-knockout (Cftr¡/¡) mice used are homozygous for the S489X-CFTR mutation (B6;129-Cftrtm1Unc ) (Snouwaert et al. 1992). Login to comment
38 The S489X mouse third generation backcross to C57BL/6 are maintained at the French Centre of Transgenesis, Archiving and Animal Models in Orleans (MF Bertrand, CDTA, Orléans, France) by random mating of heterozygous mice and are provided with their wild-type littermates (Cftr+/+) at 21-25 days of age. Login to comment

PMID: 20799947 [PubMed] Gould NS et al: "Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury."

No. Sentence Comment

48 C57BL/6J congenic gut-corrected Cftr KO-Tg mice that possess a S489X truncated mutation in the murine equivalent to CFTR and have intestinal specific expression of normal human Cftr driven by the fatty acid binding promoter were originally obtained from Case Western Reserve University`s CF Animal Core, as previously described [18]. Login to comment
187 The CFTR KO mice utilized in this study possess the S489X truncation mutation resulting in a non functional CFTR. Login to comment

PMID: 21220348 [PubMed] Radtke AL et al: "Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome."

No. Sentence Comment

310 Eckman EA, Cotton CU, Kube DM, Davis PB (1995) Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

PMID: 21547726 [PubMed] Ziady AG et al: "Methods for evaluating inflammation in cystic fibrosis."

No. Sentence Comment

598 For example, when bred into the C57BL/6 background R117H mutant mice exhibit CF defects similar in magnitude to those observed in mice bearing the F508del or S489X mutation. Login to comment

PMID: 21625623 [PubMed] Xu WM et al: "Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia."

No. Sentence Comment

29 Results Impaired spermatogenesis in CF mice models To investigate possible involvement of CFTR in spermatogenesis, we used a CFTR knockout (Cftrtm1Unc , also referred as S489X) mice. Login to comment
30 Since most homozygous S489X CF mice die at a young age or less frequently available, heterozygous mice were used for quantitative measurement. Login to comment
31 Morphological study showed that testis tissue size and weight were significantly lower in heterozygous S489X CF mice than that of wild-type control (Fig. 1A). Login to comment
32 Daily sperm production (DSP), which is often used to evaluate the spemiogenesis in the testis [21], was significantly reduced in heterozygous S489X CF mice compared with wide-type control (Fig. 1B). Login to comment
33 The sperm number recovered from the epididymis of the heterozygous S489X CF mice was also significantly lower than that of wide-type control (Fig. 1C). Login to comment
34 H&E staining showed slight decrease in diameter of seminiferous tubules (Fig. 1D,E) and slight cytoplasmic shrinkage of spermatocytes and round spermatids (Fig. S1) in S489X CF mice. Login to comment
35 Realtime PCR results showed significant reduction of Protamine-2, a specific marker of spermatid and spermatozoa, in heterozygous and homozygous S489X CF mice at mRNA level (Fig. 1F). Login to comment
36 Immunofluorescence results further demonstrated the down-regulation of Protamine-2 in S489X CF mice at protein level (Fig. 1G). Login to comment
37 CREM, a spermatid-specific transcription factor, was also down-regulated in the homozygous S489X CF mice compared to wild-type as indicated by Western blot and immunofluorescent staining respectively (Fig. 1H&J), suggesting defect in spermatogenesis in CF mice. Login to comment
38 Interestingly, immunofluorescence result showed that cAMP-responsive element binding protein (CREB), an important transcription factor in Sertoli cells during spermatogenesis, was decreased in the Sertoli cells of homozygous S489X CF mice, as compared to heterozygotes and wild-type (Fig. 1I). Login to comment
39 Western blot result also showed downregulation of phosphorylated and total CREB in homozygous S489X CF mice compared to wild-type and heterozygotes (Fig.1K). Login to comment
70 (A) Significantly reduced testis size and weight seen in heterozygous S489X mice compared with wild-type mice (n = 3; *:p,0.05). Login to comment
71 (B) Reduced daily sperm production (DSP) values retrieved from heterozygous S489X testes (n = 4; *:p,0.05). Login to comment
72 (C) Reduced sperm numbers recovered from the epididymis of heterozygous S489X mice (+/+ n = 6, +/2 n = 4; *:p,0.05). Login to comment
73 (D) H&E staining of the cross sections of wild2type, heterozygous and homozygous S489X CF mice testes. Login to comment
78 (F) Realtime PCR of protamine-2 in S489X CF mice testes. Login to comment
79 Protamine-2 mRNA level is significantly lowered in heterozygous and homozygous S489X CF mice compared to their wildtype littermates. Login to comment
80 (G) Immunofluorescent staining of Protamine-2 in +/+, +/2 and 2/2 S489X CF mice testes (stage VII-VIII). Login to comment
111 (H) Immunofluorescent staining of CREM in S489X CF mice testes (stage VIII-I). Login to comment
115 (I) Immunofluorescent staining of CREB in S489X CF mice testes shows stronger immunoactivity in +/+ and +/2 mice Sertoli cell compared to that of 2/2. Login to comment
117 (J) Western blot analysis of CREM expression in S489X CF testes. Login to comment
119 (K) Western blot analysis of CREB expression in S489X CF testes. Login to comment
126 Materials and Methods Animal and cell culture cftrtm1Unc (S489X) mice [45] were from Jackson`s laboratory and maintained in LASEC of CUHK. Login to comment

PMID: 21658634 [PubMed] Wilke M et al: "Mouse models of cystic fibrosis: phenotypic analysis and research applications."

No. Sentence Comment

67 Unfortunately, there is no Table 1 CFTR mutant mice Mouse Mutation Cftr mRNA Genetic Survival to Body wt Contact References background maturity Null mutations Cftrtm1Unc S489X Ex10 R Not detectable* C57Bl/6 <5% 10-25% reduction BH Koller/Jax Labs [113,158] Cftrtm1Cam R487X Ex10 R Not detectable 129S6/Sv/Ev <5% 20% reduction WH Colledge [159] Cftrtm1Hsc M1X Ex1 R Not detectable CD1 x 129 25% Delayed LC Tsui [160] Cftrtm3Bay Ex2 R Not detectable C57Bl/6 x 129 40% Reduced AT Beaudet [161] Cftrtm3Uth Y122X Ex4 R Not detectable C57Bl/6 25% 25-50% reduction M Capecchi/PB Davis [113,162] Hypomorphic mutations Cftrtm1Hgu ** Ex10 I 10% of wt MF1 x 129 90% No reduction J Dorin [113] Cftrtm1Bay Ex3 I <2% wt C57Bl/6 x 129 40% 70% reduction AL Beaudet [163] F508del mutations Cftrtm2Cam F508del R 30% of wt 129S6/Sv/Ev <5% 10-20% reduction WH Colledge [164] Cftrtm1Kth F508del R Low in intestine C57Bl/6 x 129 40% 10-50% reduction KR Thomas/Jax labs [165] Cftrtm1Eur F508del (H&R) Normal levels FVB/129; FVB 90% 10-20% reduction BJ Scholte [9] C57Bl/6 Other point mutations Cftrtm2Hgu G551D R 50% of wt CD1/129 65% 30-50% reduction J Dorin [11] Cftrtm3Hgu G480C (H&R) Normal levels C57Bl/6/129 Normal No reduction J Dorin [166] Cftrtm2Uth R117H R 5-20% of wt C57/Bl6 95% 10-25% reduction M Capecchi/PB Davis [113,162] Transgenes Mouse Transgene Promoter Expression Phenotype References Tg(FABPCFTR) CFTR Rat intestinal fatty acid Intestinal villus epithelia Rescue of CF intestinal pathology [167] binding protein Tg(CCSPScnn1b) Scnn1b Clara cell secretory Airway surface epithelia Na+ hyperabsorption [13] protein (CCSP) Reduced airway surface fluid volume Mucus accumulation, CF-like lung disease; 40% survival. Login to comment

PMID: 21712022 [PubMed] Fiorotto R et al: "Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4-NF-kappaB-Mediated Inflammatory Response in Mice."

No. Sentence Comment

47 Congenic B6.129P2-Cftrtm1Unc mice, (harbouring a mutation, S489X, blocking CFTR transcription), and their wild type littermates were used as an accepted model for the human CF disease13 . Login to comment
25 Animals and Experimental Protocol Experiments were performed according to protocols approved by the Yale University Institutional Animal Care and Use Committee. Congenic B6.129P2-Cftrtm1Unc mice (harboring a mutation, S489X, blocking CFTR transcription) and their WT littermates were used as an accepted model for the human CF disease.13 Genotyping for each mouse and breeding were performed as described14,15 (see Supplementary Materials and Methods). Login to comment

PMID: 9843919 [PubMed] Cressman VL et al: "The relationship of chronic mucin secretion to airway disease in normal and CFTR-deficient mice."

No. Sentence Comment

18 We used this technique to create a mouse line (Cftrm1Unc ) in which the Cftr gene was inactivated by introduction of an in-frame stop codon (termed allele S489X) that resulted in the production of a truncated gene product similar to that seen in some human CF patients (4). Login to comment
115 Results Induction of Goblet Cell Hyperplasia in BALB/c, C57BL/6, DBA/2, and 129/SvEv Strains The genetic background on which the S489X mutation is carried consists of a mixture of the BALB/c, C57BL/6, DBA/2, and 129/SvEv mouse strains. Login to comment

PMID: 22266045 [PubMed] Gould NS et al: "CFTR is the primary known apical glutathione transporter involved in cigarette smoke-induced adaptive responses in the lung."

No. Sentence Comment

39 Male CFTR KO mice that possess the S489X truncation mutation with gut-corrected recombinant human CFTR were obtained from our in-house colony as previously reported [6]. Login to comment

PMID: 22552906 [PubMed] Tang XX et al: "Lymphocyte CFTR promotes epithelial bicarbonate secretion for bacterial killing."

No. Sentence Comment

38 Materials and Methods Animals The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment

PMID: 22658665 [PubMed] Ooi CY et al: "Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis."

No. Sentence Comment

855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

PMID: 22170719 [PubMed] Chen H et al: "Impaired CFTR-dependent amplification of FSH-stimulated estrogen production in cystic fibrosis and PCOS."

No. Sentence Comment

19 In line with the observation in CF patients, CF mouse models with S489X (17) and DF508 mutation (18) in CFTR also display ovarian disorders with delayed puberty, reduced ovarian size and weight, decreased ovulated oocyte number, longer estrous cycle length, or even the absence of estrous cycle (19). Login to comment
23 Materials and Methods Animals Female ICR (Institute for Cancer Research) mice, cftrtm1Unc (S489X) mice (17), cftrtm1Kth (DF508) mice (18), and Sprague-Dawley (SD) rats were maintained in the Laboratory Animal Service Center, the Chinese University of Hong Kong. Login to comment
146 A, Immunofluorescent staining of CREB and phospho-CREB in wild-type (WT) and homozygous CFTR knockout (S489X) mice ovaries. Login to comment
183 The expression levels of CREB and phospho-CREB were reduced in the ovaries of homozygous CFTR knockout (S489X) mice compared with the wild-type control, as shown by immunofluorescent staining (Fig. 4A), suggesting that the cAMP-CREB signaling pathway is impaired in CFTR knockout mice. Login to comment

PMID: 22005837 [PubMed] Valque H et al: "Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice."

No. Sentence Comment

53 The Cftr-knockout mice used are homozygous for the S489X-CFTR mutation (B6;129-Cftr tm1Unc) (Snouwaert et al. 1992). Login to comment
54 The S489X mouse third-generation backcross to C57BL/6 were maintained at the French Centre of Transgenesis, Archiving and Animal Models in Orléans (MF Bertrand, CDTA, Orléans, France) by random mating of heterozygous mice and were obtained with their wild-type littermates (Freedman et al. 1999) at 21-25 days of age. Cftr¡/¡ mice may die shortly after weaning because of intestinal obstruction, and both Cftr¡/¡ and Cftr+/+ mice received polyethylene glycol 4000 added to the drinking water (44.4 g/l) to increase the survival of the Cftr¡/¡ mice. Login to comment

PMID: 18687795 [PubMed] Audrezet MP et al: "Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene."

No. Sentence Comment

97 Differences in melting plots obtained for a heterozygous F508del and two compounds heterozygous (F508del/S492F and F508del/S489X). Login to comment

PMID: 15033444 [PubMed] Perez-Cornejo P et al: "Regulation of Ca(2+)-activated chloride channels by cAMP and CFTR in parotid acinar cells."

No. Sentence Comment

109 To directly test this idea, we used a CFTR knockout model mice that express the S489X mutation, which results in a nonfunctional truncated channel protein [24]. Login to comment
108 To directly test this idea, we used a CFTR knockout model mice that express the S489X mutation, which results in a nonfunctional truncated channel protein [24]. Login to comment

PMID: 10923036 [PubMed] Claustres M et al: "Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France."

No. Sentence Comment

105 d G149R, S489X, S492F, S549R, 1898+1G>A, 2622+1G>A, G970R, R1066H, W1204X, 3850-1G>A, Q1313X. Login to comment

PMID: 9851885 [PubMed] Morrow SL et al: "Modification of development by the CFTR gene in utero."

No. Sentence Comment

22 A recombinant adenovirus containing human cftr (Av1CF2 [10]) was used for in utero gene transfer into the S489X cftr knockout mouse (C57Bl/ 1 To whom correspondence should be addressed at Ochsner Medical Foundation, Department of Molecular Genetics, 1516 Jefferson Highway, New Orleans, LA 70121. Login to comment
29 S489X cftr (-/-) mice develop a lethal intestinal obstruction resulting in less than 5% survival to adulthood (Ͼ40 days) (11). Login to comment
28 S489X cftr (2/2) mice develop a lethal intestinal obstruction resulting in less than 5% survival to adulthood (.40 days) (11). Login to comment

PMID: 9573345 [PubMed] Xu Z et al: "In-frame elimination of exon 10 in Cftrtm1Unc CF mice."

No. Sentence Comment

45 Tissue harvest pathway in mouse epithelial cells that may compensate for the alteration in CFTR (Clarke et al., 1992a,b; Alton The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment
46 Tissue harvest pathway in mouse epithelial cells that may compensate for the alteration in CFTR (Clarke et al., 1992a,b; Alton The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment

PMID: 9541118 [PubMed] De Braekeleer M et al: "Is meconium ileus genetically determined or associated with a more severe evolution of cystic fibrosis?"

No. Sentence Comment

16 Although the A455E mutation is Table 1 Distribution of meconium ileus among CFTR genotypes in Saguenay Lac-Saint-Jean No of CF Proportion No of CFpatients Proportion Proportion ofMI Genotypes patients (%) with meconium ileus (%) among genotypes AF508/AF508 52 39.4 5 26.3 9.6 AF508/621+G-*T 34 25.8 9 47.4 26.5 AF508/A455E 14 10.6 0 0.0 0.0 621+1G-*T/A455E 8 6.1 0 0.0 0.0 621+1G-*T/G85E 2 1.5 1 5.3 50.0 621+1G-*T/Y1092X 1 0.8 0 0.0 0.0 AF508/Y1092X 4 3.0 1 5.3 25.0 A455E/R117C 1 0.8 0 0.0 0.0 AF508/I148T 2 1.5 0 0.0 0.0 621+1G-*T/ 4 3.0 0 0.0 0.0 711 +1G-*T 621+1G-4T/S489X 1 0.8 0 0.0 0.0 AF508/Q890X 1 0.8 1 5.3 100.0 621+1G->T/ 6 4.5 2 10.5 33.3 621+1G-sT AF508/unknown 1 0.8 1 5.3 100.0 Unknown/unknown 1 0.8 0 0.0 0.0 Table 2 Main clinicalfindings in patients with meconium ileus With MI Without MI p value No of patients 18 18 Sex (M/IF) 6/12 6/12 No of patients alive 16 17 Mean age (SD) 16.75 (9.7) 16.70 (7.9) p=0.99 Mean birth weight (SD) 3.24 (0.40) 3.02 (0.47) p=O.18 Mean birth height (SD) 50.0 (2.27) 50.0 (2.58) p=0.86 Currentweightcentile (SD) 26.7 (24.5) 14.1 (18.0) p=0.06 Current height centile (SD) 29.9 (25.1) 20.6 (25.6) p=0.33 Sweat chloride concentration (mEq/l) 105.9 (6.5) 101.1 (9.8) p=O.12 Mean FVC (SD) 89.7 (24.4) 93.0 (17.0) p=0.75 Mean FEV (SD) 73.1 (23.9) 75.4 (18.7) p=0.81 Mean Shwachman score (SD) 82.8 (11.8) 79.2 (12.6) p=0.36 Colonisation with Pseudomonas aeruginosa 13 14 p=0.70 Staphyloccoccus aureus 16 17 p=0.55 Haemophilus influenzae 13 14 p=0.70 Pseudomonas maltophilia 4 6 p=0.46 Pseudomonas cepacia 0 1 Pancreatic insufficiency 18 18 DIOS 7 1 p=0.016 Rectal prolapse 1 2 p=0.55 Recurrent abdominal pain 6 1 p=0.035 Diabetes mellitus 5 0 p=0.016 Liver complications 3* 0 p=0.07 Nasal polyposis 6 6 p=1.00 DIOS=distal intestinal obstruction syndrome. Login to comment

PMID: 9466536 [PubMed] Angel J et al: "Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea."

No. Sentence Comment

14 They were produced by introducing an in-frame stop to be capable of inducing a calcium-mediated increase in apical codon in the coding sequence of exon 10 at aa 489 (S489X) [7]. Login to comment
15 They were produced by introducing an in-frame stop to be capable of inducing a calcium-mediated increase in apical codon in the coding sequence of exon 10 at aa 489 (S489X) [7]. Login to comment

PMID: 9130964 [PubMed] Alton E et al: "Gene therapy for cystic fibrosis."

No. Sentence Comment

12 2 Eckman EA, Cotton CU, Kube DM, Davis PB. Dietary changes improve survival of Cftr S489X homozygous mutant mouse. Login to comment
13 Dietary changes improve survival of Cftr S489X homozygous mutant mouse. Login to comment

PMID: 9222762 [PubMed] Jordanova A et al: "SSCP analysis: a blind sensitivity trial."

No. Sentence Comment

22 List of Mutations Included in the Experiment and Original Method of Detection Used by the Referring Laboratory Referring Probe Original method laboratory no.a Mutation Exon of detection Original SSCP conditions Institut de 1 1677delTA 10 Heteroduplexes Recerca 1 1859G/C 12 DDGE Oncologica, 3 W1282X 20 SSCPb 6% 19:1 (AA:bisAA) 4°C 5h 30W Department 4 delF508 10 Heteroduplexes de Genetica 4 Q1313X 20 SSCPb 6% 19:1 (AA:bisAA) 4°C 5h 30W Molecular, 5 1609delCA 10 SSCPb 6% 19:1 (AA:bisAA) RT 28h 10W10% glycerol Barcelona, 7 T582R 12 DGGE Spain 8 1898+3G→A ivs 12 DGGE Molecular 910085 1161delC 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Genetics 860176 1138insG 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Laboratory, 930215 1154insTC 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Royal 930838 delF508 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Manchester 930127 delI507 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Children`s 931205 Q493X 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Hospital, 900592 V520F 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm UK G12984 S489X 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 910143 G551D 11 ARMS 930274 S549N 11 SSCP/Heteroduplexes 10% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 920132 1811+1G→C ivs 11 SSCP/Heteroduplexes 10% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 930140 1898+1G→A ivs 12 SSCP/Heteroduplexes 930334 W1282X 20 SSCP/Heteroduplexes 7.25% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 140735 3850-1G→A 20 SSCP/Heteroduplexes 7.25% 49:1 (AA:bisAA) 4°C 20 h 10 V/cm Laboratoire 293 G551D 11 SSCPb 5% 19:1 (AA:bisAA) 4°C 5 h 50W and de Biochimie 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol Genetique, 324 S549R 11 ASO Hybridization Centre 649 1898+1G→A ivs 12 DGGE Hospitalier 583 E585X 12 DGGE Universitaire 710 L967S 15 DGGE Montpellier, 325 S945L 15 SSCPb 5% 19:1 (AA:bisAA) 4° 5h 50W and France 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 473 N1303H 21 SSCPb 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 216 300delA 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 287 394delTT 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 559 R74W 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 237 P67L 3 DGGE 1023 R75X 3 DGGE 885 1215delG 7 DGGE 113 Y122X 4 DGGE, SSCP 356 621+1G→T ivs 4 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 709 621+2T→G ivs 4 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 802 I148T 4 DGGE 1016 Q98R 4 DGGE V75 R117H 4 SSCP 5% 19:1 (AA:bisAA) 4°C 5 h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol a Identification numbers given by referring laboratories. Login to comment
57 Type of Mutations Detected by SSCP Analysis in This Study Type of mutation Mutation Mutation characteristics Detected by SSCP analysis Deletions 1677delTA deletion of TA from 1677 Yes delF508 deletion of 3 bp from 1655 Yes delI507 deletion of 3 bp from 1648 Yes 1609delCA deletion of CA from 1609 Yes 1161delC deletion of C at 1161 Yes 300delA deletion of A at 300 Yes 394delTT deletion of TT from 394 Yes 1215delG deletion of G at 1215 No Insertions 1138insG insertion of G after 1138 Yes 1154insTC insertion of TC after 1154 Yes Base 1859G/C Yes substitutions W1282X G→A at 3978 Yes Q1313X C→T at 4069 Yes T582R C→G at 1877 Yes 1898+3G→A A→G at 1898+3 Yes Q493X C→T at 1609 Yes V520F G→T at 1690 Yes S489X C→A at 1598 Yes G551D G→A at 1784 No S549N G→A at 1778 Yes 1811+1G→C G→C at 1811+1 Yese 1898+1G→A G→A at 1898 Yes 3850-1G→A G→A at 3850-1 Yes S549R T→G at 1779 Yes E585X G→T at 1885 Yes L967S C→T at 2966 Yes S945L C→T at 2966 No N1303H A→C at 4039 Yes R74W C→T at 352 Yes P67L C→T at 332 Yes R75X C→T at 355 Yes Y122X T→A at 498 No 621+1G→T G→T at 621+1 No 621+2T→G T→G at 621+2 No I148T T→C at 575 Yes Q98R A→G at 425 Yes R117H G→A at 482 Yes FIGURE 1. Login to comment

PMID: 7491981 [PubMed] Eckman EA et al: "Dietary changes improve survival of CFTR S489X homozygous mutant mouse."

No. Sentence Comment

0 269:L625-L630, 1995. ;Am J Physiol Lung Cell Mol Physiol E. A. Eckman, C. U. Cotton, D. M. Kube and P. B. Davis homozygous mutant mouse Dietary changes improve survival of CFTR S489X You might find this additional info useful... 25 other HighWire-hosted articles:This article has been cited by http://ajplung.physiology.org/content/269/5/L625#cited-by including high resolution figures, can be found at:Updated information and services http://ajplung.physiology.org/content/269/5/L625.full can be found at:Molecular Physiology American Journal of Physiology - Lung Cellular andaboutAdditional material and information http://www.the-aps.org/publications/ajplung This information is current as of October 25, 2012. Login to comment
6 It is published 12 times a year (monthly) by the American covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of publishes original researchAmerican Journal of Physiology - Lung Cellular and Molecular Physiology Dietary changes improve survival of CFTR S489X homozygous mutant mouse ELIZABETH A. ECKMAN, CALVIN U. COTTON, DIANNE M. KUBE, AND PAMELA B. DAVIS Departments of Pediatrics, Molecular Biology and Microbiology, and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 E&man, Elizabeth A., Calvin U. Cotton, Dianne M. Kube, and Pamela B. Davis. Login to comment
7 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment
12 13): L625-L630,1995.- Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Login to comment
24 Snouwaert et al. [lo; University of North Carolina (UNC)] and Ratcliff et al. (7; University of Cambridge) designed targeting constructs to replace a portion of the CFTR gene with two copies of the neomycin resistance gene (S489X mutation) or a hypoxanthine phosphoribosyl transferase (HPRT) mini-gene, respectively. Login to comment
40 When S489X CFTR (-/-) mice are fed Peptamen complete elemental liquid diet and bedded on corn cob pellets instead of the traditional wood shavings, 88% of these animals which are alive at 10 days of age survive to maturity. Login to comment
43 UNC mice heterozygous for the S489X mutation (CFTR + /-> were purchased from the Jackson Laboratories 1040-0605/95 $3.00 Copyright o 1995 the American Physiological Society L625 (Bar Harbor, ME). Login to comment
54 Primers III and IV were used to amplify the S489X neo-disrupted allele. Login to comment
57 Both wild-type CFTR and S489X alleles were amplified in a single reaction. Login to comment
60 Expected product sizes were N 500 base pairs (bp) for the wild-type allele (primers I and II) and 650 bp for the S489X allele (primers III and IV). Login to comment
129 DISCUSSION The UNC S489X homozygous CF mice have a severe phenotype resulting from complete inactivation of the murine CFTR gene. Login to comment
148 Indeed, a few of the S489X homozygous mice placed on this regimen actually succumbed to intestinal obstruction, so it is possible that the survivors suffered intermittent bouts of this complication and consequently were runts. Login to comment
151 S489X CF knockout mice transgenic for human CFTR under control of the rat intestinal fatty acid-binding gene promoter express CFTR predominantly, but not exclusively, in the intestine and are protected against death from intestinal obstruction even without special diet or Forskolin JI + CFTR (+/&) Solid Food -O- CFTR (+/&) Peptamen +I+ CFTR (4) Peptamen -6 4 -2 0 2 4 6 8 10 Time (min) Fig. 4. Login to comment

PMID: 7507247 [PubMed] Clarke LL et al: "Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice."

No. Sentence Comment

18 Early in life, Cftr(-/-) mice [mice homozygous for the Ser-489 to Xaa (S489X) mutation] exhibit severe intestinal disease and little pulmonary disease, which resembles the progression in these organs in CF patients, including those with chain-termination mutations similar to Cftr(-/-) mice (13). Login to comment

PMID: 11161838 [PubMed] Mailleau C et al: "Glycoconjugate metabolism in a cystic fibrosis knockout mouse model."

No. Sentence Comment

22 The cftr gene was inactivated by introduction of an in-frame stop codon (the allele S489X) resulting in a truncated gene product. Login to comment
34 Female and male 129/BC mice heterozygous for the S489X mutation were crossed to obtain homozygous S489X mice (12). Login to comment
42 For the purposes of this study wild-type mice were classified as cftraf9;/af9; , mice heterozygous for the S489X mutation as cftraf9;/afa; , and mice homozygous for the S489X mutation as cftrafa;/afa; . Login to comment

PMID: 17983806 [PubMed] Fiorotto R et al: "Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion."

No. Sentence Comment

24 The composition (in mmol/L) of perfusion buffers used was essentially as described.3,4,13 Experimental Animals Congenic B6.129P2-Cftrtm1Unc mice, which possess the S489X mutation that blocks transcription of CFTR,14 and èc;F508-CFTR (B6-129-Cftrtm1Kth) mice with targeted mutation corresponding to the èc;F508 mutation in human, were used as described.15 For further information, see the supplementary online material (see Supplementary Material online at www.gastrojournal.org). Login to comment

PMID: 18778952 [PubMed] Stalvey MS et al: "CFTR mutations impart elevated immune reactivity in a murine model of cystic fibrosis related diabetes."

No. Sentence Comment

64 Mouse strains Our experiments utilized a mixed background mouse model, originating from C57BL/6J mice with the CFTR S489X / neo insertion, as developed initially at the University of North Carolina [28]. Login to comment
69 CFTR S489X / FABP-hCFTR+/+ (hereafter, designated CFTR / ), C57BL/6J and FVB/NJ mice were housed under SPF conditions at the University of Florida Animal Care Services according to NIH guidelines, with allowances of food and water ad libitum. Login to comment

PMID: 23060053 [PubMed] Lynch SV et al: "Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota."

No. Sentence Comment

237 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

PMID: 23290341 [PubMed] Darrah RJ et al: "Ventilatory pattern and energy expenditure are altered in cystic fibrosis mice."

No. Sentence Comment

169 However, these studies were conducted in a different mouse strain (129/BC) with a different Cftr mutation (S489X) than the mice used in our study. Login to comment

PMID: 24169862 [PubMed] De Lisle RC et al: "Disrupted tight junctions in the small intestine of cystic fibrosis mice."

No. Sentence Comment

208 Am J Pathol 164:1481-1493 Eckman EA, Cotton CU, Kube DM, Davis PB (1995) Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

PMID: 25158854 [PubMed] Nedvetsky PI et al: "Parasympathetic innervation regulates tubulogenesis in the developing salivary gland."

No. Sentence Comment

260 (H) Comparison of wild-type and Cftr-deficient (S489X) salivary glands from 4-month female mice. Login to comment

PMID: 25674778 [PubMed] Baker MW et al: "Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study."

No. Sentence Comment

15 Correspondence: Mei W. Baker (mwbaker@wisc.edu) Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study Mei W. Baker, MD1,2 , Anne E. Atkins, MPH2 , Suzanne K. Cordovado, PhD3 , Miyono Hendrix, MS3 , Marie C. Earley, PhD3 and Philip M. Farrell, MD, PhD1,4 Table 1ߒ CF-causing or varying consequences mutations in the MiSeqDx IUO Cystic Fibrosis System c.1521_1523delCTT (F508del) c.2875delG (3007delG) c.54-5940_273ߙ+ߙ10250del21kb (CFTRdele2,3) c.3909C>G (N1303K) c.3752G>A (S1251N) Mutations that cause CF when combined with another CF-causing mutation c.1624G>T (G542X) c.2988ߙ+ߙ1G>A (3120ߙ+ߙ1G->A) c.3964-78_4242ߙ+ߙ577del (CFTRdele22,23) c.613C>T (P205S) c.1021T>C (S341P) c.948delT (1078delT) c.2988G>A (3120G->A) c.328G>C (D110H) c.200C>T (P67L) c.1397C>A (S466X(C>A)) c.1022_1023insTC (1154insTC) c.2989-1G>A (3121-1G->A) c.3310G>T (E1104X) c.3937C>T (Q1313X) c.1397C>G (S466X(C>G)) c.1081delT (1213delT) c.3140-26A>G (3272-26A->G) c.1753G>T (E585X) c.658C>T (Q220X) c.1466C>A (S489X) c.1116ߙ+ߙ1G>A (1248ߙ+ߙ1G->A) c.3528delC (3659delC) c.178G>T (E60X) c.115C>T (Q39X) c.1475C>T (S492F) c.1127_1128insA (1259insA) c.3659delC (3791delC) c.2464G>T (E822X) c.1477C>T (Q493X) c.1646G>A (S549N) c.1209ߙ+ߙ1G>A (1341ߙ+ߙ1G->A) c.3717ߙ+ߙ12191C>T (3849ߙ+ߙ10kbC->T) c.2491G>T (E831X) c.1573C>T (Q525X) c.1645A>C (S549R) c.1329_1330insAGAT (1461ins4) c.3744delA (3876delA) c.274G>A (E92K) c.1654C>T (Q552X) c.1647T>G (S549R) c.1393-1G>A (1525-1G->A) c.3773_3774insT (3905insT) c.274G>T (E92X) c.2668C>T (Q890X) c.2834C>T (S945L) c.1418delG (1548delG) c.262_263delTT (394delTT) c.3731G>A (G1244E) c.292C>T (Q98X) c.1013C>T (T338I) c.1545_1546delTA (1677delTA) c.3873ߙ+ߙ1G>A (4005ߙ+ߙ1G->A) c.532G>A (G178R) c.3196C>T (R1066C) c.1558G>T (V520F) c.1585-1G>A (1717-1G->A) c.3884_3885insT (4016insT) c.988G>T (G330X) c.3197G>A (R1066H) c.3266G>A (W1089X) c.1585-8G>A (1717-8G->A) c.273ߙ+ߙ1G>A (405ߙ+ߙ1G->A) c.1652G>A (G551D) c.3472C>T (R1158X) c.3611G>A (W1204X) c.1679ߙ+ߙ1.6kbA>G (1811ߙ+ߙ1.6kbA->G) c.274-1G>A (406-1G->A) c.254G>A (G85E) c.3484C>T (R1162X) c.3612G>A (W1204X) c.1680-1G>A (1812-1G->A) c.4077_4080delTGTTinsAA (4209TGTT->AA) c.2908G>C (G970R) c.349C>T (R117C) c.3846G>A (W1282X) c.1766ߙ+ߙ1G>A (1898ߙ+ߙ1G->A) c.4251delA (4382delA) c.595C>T (H199Y) c.1000C>T (R334W) c.1202G>A (W401X) c.1766ߙ+ߙ3A>G (1898ߙ+ߙ 3A->G) c.325_327delTATinsG (457TAT->G) c.1007T>A (I336K) c.1040G>A (R347H) c.1203G>A (W401X) c.2012delT (2143delT) c.442delA (574delA) c.1519_1521delATC (I507del) c.1040G>C (R347P) c.2537G>A (W846X) c.2051_2052delAAinsG (2183AA->G) c.489ߙ+ߙ1G>T (621ߙ+ߙ 1G->T) c.2128A>T (K710X) c.1055G>A (R352Q) c.3276C>A (Y1092X (C>A)) c.2052delA (2184delA) c.531delT (663delT) c.3194T>C (L1065P) c.1657C>T (R553X) c.3276C>G (Y1092X (C>G)) c.2052_2053insA (2184insA) c.579ߙ+ߙ1G>T (711ߙ+ߙ 1G->T) c.3230T>C (L1077P) c.1679G>A (R560K) c.366T>A (Y122X) c.2175_2176insA (2307insA) c.579ߙ+ߙ3A>G (711ߙ+ߙ 3A->G) c.617T>G (L206W) c.1679G>C (R560T) - c.2215delG (2347delG) c.579ߙ+ߙ5G>A (711ߙ+ߙ 5G->A) c.1400T>C (L467P) c.2125C>T (R709X) - c.2453delT (2585delT) c.580-1G>T (712-1G->T) c.2195T>G (L732X) c.223C>T (R75X) - c.2490ߙ+ߙ1G>A (2622ߙ+ߙ1G->A) c.720_741delAGGGAG AATGATGATGAAGTAC (852del22) c.2780T>C (L927P) c.2290C>T (R764X) - c.2583delT (2711delT) c.1364C>A (A455E) c.3302T>A (M1101K) c.2551C>T (R851X) - c.2657ߙ+ߙ5G>A (2789ߙ+ߙ5G->A) c.1675G>A (A559T) c.1A>G (M1V) c.3587C>G (S1196X) - Mutations/variants that were validated in this study are in bold. CF, cystic fibrosis. Table 1ߒ Continued on next page reduce carrier detection and potentially improve the positive predictive value (PPV), the NBS goals of equity and the highest possible sensitivity become more difficult to achieve. Login to comment

PMID: 26014425 [PubMed] Girardet A et al: "The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus."

No. Sentence Comment

79 (unknown) Q39X c.115C4T p.Gln39* P67L c.200C4T p.Pro67Leu R75X c.223C4T p.Arg75* 405+1G4A c.273+1G4A 406-1G4A c.274-1G4A E92X c.274G4T p.Glu92* E92K c.274G4A p.Glu92Lys Q98X c.292C4T p.Gln98* 457TAT4G c.325_327delTATinsG p.Tyr109Glyfs*4 D110H c.328G4C p.Asp110His R117C c.349C4T p.Arg117Cys Y122X c.366 T4A p.Tyr122* 574delA c.442delA p.Ile148Leufs*5 444delA c.313delA p.Ile105Serfs*2 663delT c.531delT p.Ile177Metfs*12 G178R c.532G4A p.Gly178Arg 711+3 A4G c.579+3 A4G 711+5G4A c.579+5G4A 712-1G4T c.580-1G4T H199Y c.595C4T p.His199Tyr P205S c.613C4T p.Pro205Ser L206W c.617 T4G p.Leu206Trp Q220X c.658C4T p.Gln220* 852del22 c.720_741delAGGGAGAAT GATGATGAAGTAC p.Gly241Glufs*13 1078delT c.948delT p.Phe316Leufs*12 G330X c.988G4T p.Gly330* Table 1 (Continued ) HGVS nomenclature Legacy name cDNA nucleotide name Protein name R334W c.1000C4T p.Arg334Trp I336K c.1007 T4A p.Ile336Lys T338I c.1013C4T p.Thr338Ile 1154insTC c.1021_1022dupTC p.Phe342Hisfs*28 S341P c.1021 T4C p.Ser341Pro R347H c.1040G4A p.Arg347His 1213delT c.1081delT p.Trp361Glyfs*8 1248+1G4A c.1116+1G4A 1259insA c.1130dupA p.Gln378Alafs*4 W401X(TAG) c.1202G4A p.Trp401* W401X(TGA) c.1203G4A p.Trp401* 1341+1G4A c.1209+1G4A 1461ins4 c.1329_1330insAGAT p.Ile444Argfs*3 1525-1G4A c.1393-1G4A S466X c.1397C4A or c.1397C4G p.Ser466* L467P c.1400 T4C p.Leu467Pro S489X c.1466C4A p.Ser489* S492F c.1475C4T p.Ser492Phe 1677delTA c.1545_1546delTA p.Tyr515* V520F c.1558G4T p.Val520Phe 1717-1G4A c.1585-1G4A 1717-8G4A c.1585-8G4A S549R c.1645 A4C p.Ser549Arg S549N c.1646G4A p.Ser549Asn S549R c.1647 T4G p.Ser549Arg Q552X c.1654C4T p.Gln552* A559T c.1675G4A p.Ala559Thr 1811+1.6kbA4G c.1680-886 A4G 1812-1G4A c.1680-1G4A R560K c.1679G4A p.Arg560Lys E585X c.1753G4T p.Glu585* 1898+3 A4G c.1766+3 A4G 2143delT c.2012delT p.Leu671* 2184insA c.2052_2053insA p.Gln685Thrfs*4 2184delA c.2052delA p.Lys684Asnfs*38 R709X c.2125C4T p.Arg709* K710X c.2128 A4T p.Lys710* 2307insA c.2175dupA p.Glu726Argfs*4 L732X c.2195 T4G p.Leu732* 2347delG c.2215delG p.Val739Tyrfs*16 R764X c.2290C4T p.Arg764* 2585delT c.2453delT p.Leu818Trpfs*3 E822X c.2464G4T p.Glu822* 2622+1G4A c.2490+1G4A E831X c.2491G4T p.Glu831* W846X c.2537G4A p.Trp846* W846X (2670TGG4TGA) c.2538G4A p.Trp846* R851X c.2551C4T p.Arg851* 2711delT c.2583delT p.Phe861Leufs*3 S945L c.2834C4T p.Ser945Leu 2789+2insA c.2657+2_2657+3insA Q890X c.2668C4T p.Gln890* L927P c.2780 T4C p.Leu927Pro 3007delG c.2875delG p.Ala959Hisfs*9 G970R c.2908G4C p.Gly970Arg 3120G4A c.2988G4A function variants that cause CF disease when paired together; (ii) variants that retain residual CFTR function and are compatible with milder phenotypes such as CFTR-RD; (iii) variants with no clinical consequences; and (iv) variants of unproven or uncertain clinical relevance. Login to comment

PMID: 26087176 [PubMed] Dupuis A et al: "Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene."

No. Sentence Comment

63 Canadian studies for CF modfier genes 2,492 3,153 43,432 3,596 1,788 2,230 23,397 16,023 3 716 3,438 860 15% (19%) 1,902 2,576 PIP and MIP derivation FEV1 and zBMI modeling MIP calculation following correction of MI variable 23,301 2,413 510 21% (25%) 20% (23%) 13% (15%) Total F508del/others MI prevalence uncorrected (estimated) Missing or incomplete genotype Available for analysis Canadian CF patient registry, born after 1980 US CF patient registry German CF patient registry CF patient registry, North Italy Table 1ߒ Meconium ileus prevalence scores for the most common cystic fibrosis-causing variants p. F508del/other variants Class PIP Canada, (n) MIP, (n) Canada United States Germany Italy HGVS Legacy name c.262_263delTT 394delTT I 0.38 (50) c.3472C>T R1158X I 0.37 (35) c.1558G>T V520F 0.35 (43) c.3484C>T R1162X I 0.34 (135) 0.17 (14) 0.22 (45) c.2012delT 2143delT I 0.33 (13) c.3276C>A or G Y1092X I 0.92 (13) 0.09 (12) 0.33 (55) c.3846G>A W1282X I 1.00 (13) 0.29 (13) 0.32 (442) 0.17 (20) c.1477C>T Q493X I 1.00 (11) 0.19 (11) 0.32 (102) c.3528delC 3659delC I 0.31 (139) c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 0.97 (39) 0.30 (38) 0.31 (54) c.178G>T E60X I 0.30 (66) c.1657C>T R553X I 1.00 (16) 0.28 (16) 0.30 (415) 0.24 (107) c.1585-1G>A 1717-1G>A I 1.00 (12) 0.23 (12) 0.29 (367) 0.22 (38) 0.16 (22) c.1766ߙ+ߙ1G>A 1898ߙ+ߙ1G>A 0.29 (139) c.1624G>T G542X I 0.99 (73) 0.31 (72) 0.29 (976) 0.21 (79) 0.22 (33) c.1521_1523delCTT F508del II 0.99 (1292) 0.22 (1260) 0.27 (15391) 0.21 (1910) 0.20 (230) c.1679G>C R560T II 0.27 (123) c.3744delA 3876delA 0.27 (22) c.2128A>T K710X I 0.26 (12) c.1519_1521delATC I507del II 1.00 (20) 0.21 (19) 0.25 (162) c.3909C>G N1303K II 0.98 (40) 0.13 (39) 0.25 (534) 0.23 (80) 0.14 (62) c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T I 1.00 (90) 0.24 (88) 0.25 (369) 0.21 (11) c.3266G>A W1089X I 0.25 (17) c.1675G>A A559T 0.24 (21) c.988G>T G330X 0.24 (10) c.3773_3774insT 3905insT 0.23 (78) c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 0.22 (121) c.443T>C I148T;3199del6 1.00 (15) 0.22 (15) c.2052delA 2184delA I 0.21 (89) 0.22 (10) c.2051_2052delAAinsG 2183AA>G 0.20 (73) 0.20 (42) c.948delT 1078delT 0.19 (20) c.1652G>A G551D III 0.96 (54) 0.08 (53) 0.15 (979) 0.09 (84) c.254G>A G85E 0.50 (24) 0.06 (24) 0.14 (137) 0.00 (10) c.3196C>T R1066C 0.14 (42) c.1466C>A S489X 1.00 (14) 0.14 (14) c.3808G>A D1270N 0.13 (19) c.1055G>A R352Q 0.12 (18) c.579ߙ+ߙ5G>A 711ߙ+ߙ5G>A 0.12 (30) c.2175_2176insA 2307insA 0.11 (24) c.349C>T R117C 0.10 (37) c.1040G>C R347P IV 0.18 (11) 0.19 (11) 0.10 (130) 0.02 (56) c.350G>A R117H IV 0.05 (21) 0.00 (21) 0.07 (666) 0.02 (19) c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A V 0.25 (20) 0.00 (20) 0.06 (271) 0.01 (21) c.1040G>A R347H 0.06 (55) c.2988G>A 3120G->A 0.06 (36) c.328G>C D1152H IV 0.06 (124) c.3717ߙ+ߙ12191C>T 3849ߙ+ߙ10kbC>T V 0.07 (14) 0.00 (14) 0.05 (299) 0.01 (42) 0.00 (15) c.1364C>A A455E V 0.16 (45) 0.01 (41) 0.05 (109) c.1000C>T R334W IV 0.18 (11) 0.00 (10) 0.05 (92) c.617T>G L206W 0.06 (18) 0.05 (17) 0.04 (52) c.3302T>A M1101K 0.04 (17) c.200C>T P67L V 0.07 (14) 0.00 (14) Meconium ileus prevalence (MIP) and pancreas insufficiency prevalence (PIP) scores are presented. Login to comment

PMID: 26289599 [PubMed] Jansson K et al: "Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells."

No. Sentence Comment

35 Cftrm1UNC (S489X) mice, which exhibit a null CFTR phenotype due to unstable CFTR expression at the mRNA and protein level (Takacs-Jarrett et al. 2001), were also used in this study. Login to comment

PMID: 7533607 [PubMed] Snouwaert JN et al: "A murine model of cystic fibrosis."

No. Sentence Comment

33 The resulting mutation, which we have designated S489X, should result in a truncated gene product similar to that 560 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 151 1995 seen with several types of human CF mutations. Login to comment
36 Once cell lines carrying the S489X mutation were isolated, they were used to generate mice carrying the same mutation. Login to comment
37 This was accomplished by injecting the cells carrying the S489X mutation into mouse blastocysts and introducing the injected blastocysts into the uterus of a pseudopregnant female. Login to comment
39 These animals were therefore able to pass the ES cell genome, including the S489X mutation, on to their offspring. Login to comment
40 Offspring receiving the ES cell genome from one chimeric parent were initially identified based on coat color, and those heterozygous for he S489X mutation were subsequently identified by PCR analysis of tail DNA. Login to comment
46 RESULTS Survival We reported previously that genetic analysis of the offspring of CFTR(-I-) mice indicated that there was no prenatal mortality associated with the S489X mutation, because the ratio of offspring lacking the S489X mutation to those heterozygous or homozygous for the mutation did not deviate significantly from the expected Mendelian ratio of 1:2:1. Login to comment
82 Another possibility is that the S489X mutation, because it results in a truncated CFTR protein, may result in more severe intestinal complications than the ..1.508 mutation, which is the most common cause of CF in humans. Login to comment
102 Similar to the S489X mutation in our CFTR(-1-) mice, the W1282X mutation is expected to result in a truncated CFTR protein. Login to comment
163 However, the phenotype associated with the S489X mutation also differs in obvious ways from the symptoms commonly associated with human CF. Login to comment
172 With respect to genetic influences, it is possible that since the S489X mutation is still on a heterogeneous genetic background, and since only a small percentage of CFTR( -1-) mice survive, we may have selected for genotypes that compensate to some degree for the effects of the S289X mutation. Login to comment