ABCMdb

PMID: 12226741

Du M, Jones JR, Lanier J, Keeling KM, Lindsey JR, Tousson A, Bebok Z, Whitsett JA, Dey CR, Colledge WH, Evans MJ, Sorscher EJ, Bedwell DM
Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.
J Mol Med (Berl). 2002 Sep;80(9):595-604. Epub 2002 Jul 3., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly542* M. Du · J. Lanier · K.M. Keeling · D.M. Bedwell (✉) Department of Microbiology, 1530 Third Avenue, South, The University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA e-mail: dbedwell@uab.edu Tel.: +1-205-9346593, Fax: +1-205-9755482 J.R. Jones · D.M. Bedwell Department of Human Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA J.R. Lindsey Department of Genomics and Pathobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA Z. Bebök · E.J. Sorscher Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama, USA M. Du · J.R. Jones · J. Lanier · K.M. Keeling · J.R. Lindsey A. Tousson · Z. Bebök · E.J. Sorscher · D.M. Bedwell Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA J.A. Whitsett · C.R. Dey Division of Pulmonary Biology, Children`s Hospital Medical Center, Cincinnati, Ohio, USA W.H. Colledge Department of Physiology, University of Cambridge, Cambridge, UK M.J. Evans Cardiff School of Biosciences, Cardiff University, Cardiff, UK Present address: J.R. Jones, Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA J Mol Med (2002) 80:595-604 DOI 10.1007/s00109-002-0363-1 O R I G I N A L A RT I C L E Ming Du · Julie R. Jones · Jessica Lanier Kim M. Keeling · J. Russell Lindsey Albert Tousson · Zsuzsa Bebök · Jeffrey A. Whitsett Chitta R. Dey · William H. Colledge Martin J. Evans · Eric J. Sorscher · David M. Bedwell Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene Received: 1 February 2002 / Accepted: 17 May 2002 / Published online: 3 July 2002 (c) Springer-Verlag 2002 MING DU received her Ph.D. degree in Biochemistry at Chiba University, Japan. Login to comment 11 ABCC7 p.Gly542* To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. Login to comment 13 ABCC7 p.Gly542* Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Login to comment 16 ABCC7 p.Gly542* When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo. Login to comment 17 ABCC7 p.Gly542* Keywords Aminoglycoside · Cystic fibrosis · CFTR-G542X transgene · Premature stop mutation Introduction Cystic fibrosis (CF) is the most prevalent autosomal recessive disorder in the Caucasian population, affecting 1 in 2,500 newborns. Login to comment 32 ABCC7 p.Gly542* To do this, a transgenic mouse expressing a hCFTR cDNA containing the G542X stop mutation under control of the rat intestinal fatty-acid binding protein (FABP) promoter was constructed. Login to comment 33 ABCC7 p.Gly542* We then examined the ability of aminoglycosides to induce hCFTR expression in Cftr-/- hCFTR-G542X mice. Login to comment 35 ABCC7 p.Gly542* These results provide evidence that gentamicin (and to a lesser extent, tobramycin) can suppress the hCFTR-G542X mutation in vivo. Login to comment 36 ABCC7 p.Gly542* Materials and methods Plasmid construction The plasmid containing hCFTR-G542X under control of the rat FABP promoter was derived from a FABP-hCFTR-WT plasmid [14]. Login to comment 37 ABCC7 p.Gly542* ABCC7 p.Gly542* The G542X premature stop mutation was introduced by the direct exchange of a 3043 bp BspE1/NcoI fragment from pDB436, yielding the FABP-CFTR-G542X plasmid pDB488. Login to comment 38 ABCC7 p.Gly542* In addition to the G542X mutation, this plasmid contains an additional SalI restriction site that was introduced at codon 764 of the CFTR cDNA. Login to comment 40 ABCC7 p.Gly542* Generation of transgenic mice A 1.2 kb DNA fragment containing the rat intestinal fatty-acid binding protein (FABP) promoter (kindly provided by Jeffrey Gordon, Washington University, St. Louis) was used to direct expression of the human CFTR cDNA containing the G542X mutation to the intestinal epithelial cells of mice using methods previously described [14]. Login to comment 41 ABCC7 p.Gly542* The chimeric FABP-hCFTR-G542X gene construct was microinjected into fertilized oocytes. Login to comment 43 ABCC7 p.Gly542* The FABP-hCFTR-G542X transgene was detected by Southern analysis in founder mice using a 2.54 kb BamHI fragment from the hCFTR gene as a probe. Login to comment 45 ABCC7 p.Gly542* Animals that expressed the FABP-hCFTR-G542X transgene were then bred with heterozygous Cftr+/Cftrtm1Cam mice (hereafter referred to as Cftr+/- mice). Login to comment 48 ABCC7 p.Gly542* ABCC7 p.Gly542* Animals that contained single copies of both the Cftrtm1Cam allele and the FABP-hCFTR-G542X transgene were interbred until the NeoR marker was eliminated and the progeny were homozygous for the FABP-hCFTR-G542X transgene. Login to comment 49 ABCC7 p.Gly542* Cftr+/- mice that were homozygous for the FABP-hCFTR-G542X transgene were then intercrossed to generate the desired Cftr-/- progeny carrying the transgene. Login to comment 53 ABCC7 p.Gly542* Amplification of the FABP-hCFTR-G542X transgene DNA was carried out using the primers DB985 (5'-CAAGATAGAA AGAGGACAGT TGTT-3') and DB986 (5'-TTGAGGGTTG ACATAGGTGC TTGAA-3'). Login to comment 54 ABCC7 p.Gly542* This primer pair generated a 1557 bp fragment containing both the G542X mutation and the new SalI site. Login to comment 57 ABCC7 p.Gly542* ABCC7 p.Gly542* mRNA expression in intestinal tissue To determine whether the FABP-CFTR-G542X transgene was expressed, mRNA was isolated from intestinal tissues of FABP-hCFTR-G542X or FABP-hCFTR-WT transgenic mice. Login to comment 59 ABCC7 p.Gly542* The hCFTR-G542X PCR product was digested with the restriction enzyme SalI to demonstrate that the predicted 954 and 603 bp fragments were produced. Login to comment 63 ABCC7 p.Gly542* Aminoglycoside treatment Aminoglycoside treatment of homozygous Cftr-/- mice carrying the FABP-hCFTR-G542X transgene was initiated 16 days after birth (1 week before weaning). Login to comment 78 ABCC7 p.Gly542* In control experiments, glibenclamide was found to block the forskolin-activated Isc in Cftr+/- mice carrying the hCFTR-G542X transgene. Login to comment 93 ABCC7 p.Gly542* In vitro transcription/translation A modified form of a readthrough reporter system previously used to monitor the suppression of stop mutations by aminoglycosides [10] was used to determine whether the aminoglycosides gentamicin and tobramycin could suppress the CFTR-G542X mutation in an in vitro rabbit reticulocyte lysate translation system (Promega TNT system). Login to comment 96 ABCC7 p.Gly542* In this study, the readthrough cassette contained the CFTR-G542X premature stop mutation and six upstream and downstream codons from the hCFTR gene. Login to comment 100 ABCC7 p.Gly542* Efficient translation termination at the G542X stop mutation produced a 25 kDa protein, while suppression of the stop codon resulted in the synthesis of a 35 kDa protein. Login to comment 102 ABCC7 p.Gly542* Suppression of the G542X mutation was expressed as percent readthrough, which was calculated as the [35 kDa protein / (25 kDa + 35 kDa proteins)] × 100. Login to comment 103 ABCC7 p.Gly542* Results Genetic characterization of a Cftr-/- mouse expressing the hCFTR-G542X transgene The objective of this study was to determine whether the aminoglycosides gentamicin or tobramycin can suppress a hCFTR premature stop mutation in an animal model. Login to comment 105 ABCC7 p.Gly542* As a result, we bred FABP-hCFTR-G542X mice with Cftr+/- mice that carried the HPRT gene inserted in exon 10 of the mouse Cftr gene [4, 15]. Login to comment 106 ABCC7 p.Gly542* Male and female Cftr+/- mice that were homozygous for the FABP-hCFTR-G542X transgene were produced and used as breeders for all subsequent experiments. Login to comment 107 ABCC7 p.Gly542* From these breeder pairs, offspring homozygous for both the Cftrtm1Cam allele and the FABP-hCFTR-G542X transgene were obtained at a frequency of 25%. Login to comment 108 ABCC7 p.Gly542* ABCC7 p.Gly542* To confirm the presence of the FABP-hCFTR-G542X transgene in these animals, a PCR reaction with hCFTR-specific primers was carried out using genomic DNA isolated from both the FABP-hCFTR-G542X and FABP-hCFTR-WT transgenic mice. Login to comment 110 ABCC7 p.Gly542* As a unique indentifier, the hCFTR-G542X transgene also contained a new SalI restriction site polymorphism that does not alter the amino acid sequence of the hCFTR protein. Login to comment 111 ABCC7 p.Gly542* Digestion of the PCR products with SalI produced the expected restriction fragments of 954 and 603 bp from the PCR product from the hCFTR-G542X mouse, but not from the hCFTR-WT mouse. Login to comment 112 ABCC7 p.Gly542* This result confirmed the presence of the FABP-hCFTR-G542X transgene. Login to comment 113 ABCC7 p.Gly542* We next analyzed the expression of the FABP-hCFTR-G542X transgene. Login to comment 114 ABCC7 p.Gly542* Since the FABP promoter is expressed predominantly in intestine [14], mRNA was isolated from intestinal tissues from either hCFTR-G542X or hCFTR-WT transgenic mice. Login to comment 116 ABCC7 p.Gly542* As can be seen in Fig. 1, a 1,557 bp fragment could be detected in both the hCFTR-G542X and hCFTR-WT transgenic mice. Login to comment 117 ABCC7 p.Gly542* In addition, digestion of the cDNA fragment generated by RT-PCR with SalI site again confirmed that a SalI site was present exclusively in the mouse line carrying the hCFTR-G542X transgene. Login to comment 118 ABCC7 p.Gly542* ABCC7 p.Gly542* These results confirmed that the FABP-hCFTR-G542X construct is expressed in intestinal tissue from the FABP-hCFTR-G542X mice. Login to comment 119 ABCC7 p.Gly542* ABCC7 p.Gly542* Pathologic characterization of Cftr-/- mice expressing the hCFTR-G542X transgene Mice homozygous for both the Cftr-/- allele and the FABP-hCFTR-G542X transgene were generally smaller than their heterozygous littermates, as observed in various Cftr-/- mouse models [19]. Login to comment 121 ABCC7 p.Gly542* While the survival of the Cftr-/- hCFTR-G542X mice was generally high during the first few weeks after birth, we found that most of these animals died in the days following weaning. Login to comment 123 ABCC7 p.Gly542* To determine the primary cause of death of the Cftr-/- hCFTR-G542X mice, a comprehensive histopathologic examination was carried out on several animals. Login to comment 124 ABCC7 p.Gly542* In particular, our attention focused on comparing healthy and sick Cftr-/- hCFTR-G542X mice in the days following weaning. Login to comment 127 ABCC7 p.Gly542* Some of these findings are il- 598 Fig. 1A, B Generation of Cftr-/- FABP-hCFTR-G542X mice. Login to comment 128 ABCC7 p.Gly542* A Schematic diagram of the FABP-hCFTR-G542X construct. Login to comment 129 ABCC7 p.Gly542* ABCC7 p.Gly542* B PCR from genomic DNA showing the presence of the hCFTR-G542X transgene (left) and an RT-PCR showing that the hCFTR-G542X transgene is expressed in intestinal tissues (right). Login to comment 131 ABCC7 p.Gly542* A similar analysis of Cftr+/+ FABP-hCFTR-G542X littermates found that all tissues were normal. Login to comment 132 ABCC7 p.Gly542* These results confirmed that the cftr-/- FABP-hCFTR-G542X mice, like other Cftr-/-mouse models, frequently experience intestinal blockage. Login to comment 133 ABCC7 p.Gly542* However, little or no intestinal pathology was observed in Cftr-/- hCFTR-G542X mice that were not ill, other than a minimal retention of mucus in the crypts of the small and large intestine (data not shown). Login to comment 135 ABCC7 p.Gly542* In preliminary experiments, we found that the subcutaneous administration of gentamicin at a similar dose was well tolerated in the FABP-hCFTR-G542X mice. Login to comment 139 ABCC7 p.Gly542* ABCC7 p.Gly542* Effect of aminoglycoside treatment on the survival of Cftr-/- hCFTR-G542X mice We first asked whether the subcutaneous administration of the aminoglycosides increased the survival of Cftr-/- mice carrying the hCFTR-G542X transgene. Login to comment 141 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* As a control for any change in survival of these animals caused by antibacterial effects rather than suppression of the hCFTR-G542X stop mutation, we also treated a control group of mice 599 Fig. 2 Histopathology observed in the jejunum of: A a Cftr+/+ mouse carrying the hCFTR-G542X transgene, and B an ill Cftr-/- mouse carrying the hCFTR-G542X transgene. Login to comment 146 ABCC7 p.Gly542* Groups of both treated and untreated Cftr-/- hCFTR-G542X mice had a high rate of survival up to 20 days after birth (Fig. 4). Login to comment 154 ABCC7 p.Gly542* hCFTR activity is detected in Cftr-/- hCFTR-G542X mice following aminoglycoside treatment The CFTR protein is a cAMP-activated chloride channel that facilitates transepithelial chloride conductance following its activation by cAMP agonists. Login to comment 156 ABCC7 p.Gly542* In intestinal tissue from untreated Cftr+/- hCFTR-G542X mice, we observed a strong increase in transepithelial chloride current that ranged from 6.2 to 26.6 µA/cm2 following forskolin addition, demonstrating the presence of endogenous CFTR activity in these heterozygous animals. Login to comment 157 ABCC7 p.Gly542* ABCC7 p.Gly542* When in600 Fig. 4 Survival of homozygous Cftr-/- hCFTR-G542X mice. Login to comment 160 ABCC7 p.Gly542* A Tracing from the ileum of a heterozygous Cftr +/- mouse carrying the hCFTR-G542X (without aminoglycoside treatment). Login to comment 161 ABCC7 p.Gly542* B Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X mouse (without aminoglycoside treatment). Login to comment 162 ABCC7 p.Gly542* C Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X (following gentamicin treatment). Login to comment 163 ABCC7 p.Gly542* D Tracing from the ileum of a homozygous Cftr-/- hCFTR-G542X (following tobramycin treatment). Login to comment 167 ABCC7 p.Gly542* The "n" value represents the total number of samples assayed under each condition testinal tissue from untreated Cftr-/- hCFTR-G542X mice was examined following forskolin treatment, no change in the transepithelial chloride current was detected in 13 independent sample s (Figs. Login to comment 171 ABCC7 p.Gly542* In Cftr-/- hCFTR-G542X mice treated with gentamicin, we observed a modest increase in Isc upon forskolin addition in 6 of 15 samples. Login to comment 174 ABCC7 p.Gly542* In cftr-/- hCFTR-G542X mice treated with tobramycin, we observed an Isc increase in 3 of 11 samples following forskolin addition, which approached statistical significance (P=0.052). Login to comment 176 ABCC7 p.Gly542* These results suggest that both gentamicin and tobramycin can restore a low level of hCFTR activity in Cftr-/- hCFTR-G542X mice. Login to comment 177 ABCC7 p.Gly542* hCFTR protein can be detected in intestinal glands following aminoglycoside treatment Since our functional analysis detected the presence of cAMP-activated Isc currents in intestinal tissues of Cftr-/- hCFTR-G542X mice following aminoglycoside treatment, we next asked whether the hCFTR protein could be detected. Login to comment 179 ABCC7 p.Gly542* In gentamicin-treated Cftr -/- hCFTR-G542X 601 Fig. 7 CFTR immunofluorescence in intestinal tissues. Login to comment 180 ABCC7 p.Gly542* Samples from the duodenum of homozygous Cftr-/- hCFTR-G542X mice (harvested from untreated, gentamicin-treated, or tobramycin-treated animals) were incubated with either preimmune serum or CFTR-NBD1 serum. Login to comment 181 ABCC7 p.Gly542* Similar samples from heterozygous Cftr+/- animals that lacked the hCFTR-G542X transgene were also examined following gentamicin treatment. Login to comment 186 ABCC7 p.Gly542* Furthermore, a positive signal was not observed in tissues from gentamicin-treated Cftr -/- hCFTR-G542X mice when a pre-immune serum was used, or when the hCFTR-specific antiserum was used on samples from mice that had not been treated with gentamicin. Login to comment 187 ABCC7 p.Gly542* We also confirmed that a specific signal was not observed in Cftr+/- mice that did not carry the hCFTR-G542X transgene following gentamicin treatment. Login to comment 189 ABCC7 p.Gly542* When taken together with the functional data presented above, these results indicate that treatment of Cftr-/- hCFTR-G542X mice with either gentamicin or tobramycin can promote the synthesis of full-length, functional CFTR that localizes to the apical epithelium of the submucosal and mucosal glands of this transgenic mouse. Login to comment 193 ABCC7 p.Gly542* ABCC7 p.Gly542* To more directly determine the ability of gentamicin and tobramycin to suppress the hCFTR-G542X mutation, we constructed a readthrough reporter construct for use in an in vitro translation system that contains the hCFTR-G542X stop mutation and six flanking codons upstream and downstream of the premature stop mutation (Fig. 8). Login to comment 205 ABCC7 p.Gly542* B Example of gentamicin- and tobramycin-mediated suppression of the hCFTR-G542X stop mutation. Login to comment 206 ABCC7 p.Gly542* C Quantitation of data shown in B full-length CFTR from a transgene containing the hCFTR-G542X premature stop mutation in vivo. Login to comment 208 ABCC7 p.Gly542* To optimize our chance of detecting the appearance of hCFTR protein produced by suppression of the hCFTR-G542X mutation, we administered 34 µg/g gentamicin by subcutaneous injection a once daily. Login to comment 214 ABCC7 p.Gly542* Our results also demonstrate for the first time that tobramycin is capable of weakly suppressing the hCFTR-G542X stop mutation in vivo. Login to comment 215 ABCC7 p.Gly542* The hCFTR-G542X stop mutation is a UGAG tetranucleotide termination signal. Login to comment 219 ABCC7 p.Gly542* Both gentamicin and tobramycin were found to suppress the hCFTR-G542X stop mutation, although gentamicin appeared to mediate this effect much more efficiently than tobramycin. Login to comment 237 ABCC7 p.Gly542* In the present study, we used a similar FABP-hCFTR construct to show that aminoglycosides can suppress the hCFTR-G542X mutation. Login to comment 238 ABCC7 p.Gly542* Significantly, we found that gentamicin treatment of the FABP-hCFTR-G542X mouse resulted in the appearance of CFTR in the mucosal and submucosal glands, but not in the crypts of Lieberkuhn or the intestinal villi. Login to comment