Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.
Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.
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DOI：
10.1042/ba20080266
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发表时间：
2009-07-09
影响因子：
2.8
通讯作者：
Gambari R
中科院分区：
文献类型：
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作者：
Salvatori F;Cantale V;Breveglieri G;Zuccato C;Finotti A;Bianchi N;Borgatti M;Feriotto G;Destro F;Canella A;Breda L;Rivella S;Gambari R
无义突变，在mRNA的编码区内产生UAA、UGA和UAG终止密码子，促进过早翻译终止，并且是导致约1000个突变的主要原因。30%的遗传性疾病，包括囊性纤维化、杜氏肌营养不良和地中海贫血。例如，在β039-地中海贫血中，CAG（谷氨酰胺）密码子突变为UAG终止密码子，导致过早翻译终止和通过充分描述的NMD（无义介导的mRNA衰变）导致mRNA不稳定。为了开发一种促进翻译的方法，从而保护免受NMD，氨基糖苷类抗生素已经在携带提前终止密码子的mRNA上进行了测试。这些药物降低密码子-反密码子碱基配对的准确性，诱导提前终止密码子的核糖体通读。有趣的是，最近的论文描述了设计和生产用于抑制过早翻译终止的药物，诱导核糖体通读过早但非正常终止密码子。这些发现为开发β039-地中海贫血的药理学治疗方法带来了新的希望。在此背景下，我们开始开发β039-地中海贫血突变的细胞模型，该模型可用于筛选大量氨基糖苷类和类似分子。为此目的，我们产生了在最小LCR（基因座控制区）控制下含有β039-地中海贫血珠蛋白基因的慢病毒构建体，并使用该构建体转导K562细胞，随后亚克隆，目的是获得具有该构建体的不同整合拷贝的几个K562克隆。然后用遗传霉素（也称为G418）和其他氨基糖苷类处理这些克隆，并通过FACS分析来分析β-珠蛋白的产生。结果表明，该实验系统适用于β039-珠蛋白突变引起的β地中海贫血的校正。
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 β039-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 β039-thalassaemia. In this context, we started the development of a cellular model of the β039-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 β039-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 β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing β-thalassaemia.