title: Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.
abstract: Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0)39-globin mutation causing beta-thalassaemia.
authors.affiliation: BioPharmaNet, Department of Biochemistry and Molecular Biology, Ferrara University, 44100 Ferrara, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvatori
authors.forename: Vera
authors.initials: V
authors.lastname: Cantale
authors.forename: Giulia
authors.initials: G
authors.lastname: Breveglieri
authors.forename: Cristina
authors.initials: C
authors.lastname: Zuccato
authors.forename: Alessia
authors.initials: A
authors.lastname: Finotti
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Bianchi
authors.forename: Monica
authors.initials: M
authors.lastname: Borgatti
authors.forename: Giordana
authors.initials: G
authors.lastname: Feriotto
authors.forename: Federica
authors.initials: F
authors.lastname: Destro
authors.forename: Alessandro
authors.initials: A
authors.lastname: Canella
authors.forename: Laura
authors.initials: L
authors.lastname: Breda
authors.forename: Stefano
authors.initials: S
authors.lastname: Rivella
authors.forename: Roberto
authors.initials: R
authors.lastname: Gambari
mesh_terms: D002999:Clone Cells; D003001:Cloning, Molecular; D018389:Codon, Nonsense; D015870:Gene Expression / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D020014:K562 Cells / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D055544:beta-Globins / Q000096:biosynthesis / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics*
chemical_list: D018389:Codon, Nonsense; D005839:Gentamicins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D055544:beta-Globins; D049452:Green Fluorescent Proteins; C010680:antibiotic G 418