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首页> 外文期刊>Gene Therapy and Molecular Biology >Multiple detection of chromosomal gene correctionmediated by a RNA/DNA oligonucleotide
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Multiple detection of chromosomal gene correctionmediated by a RNA/DNA oligonucleotide

机译：RNA / DNA寡核苷酸介导的染色体基因校正的多重检测

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Chimeric RNA/DNA oligonucleotide (RDO)-mediated gene correction of a single base mutation in a gene of an eukaryotic cell is still a controversial strategy. To better define the potential and applicability of this strategy, new systems, that allow to detect RDO-mediated gene correction in the chromosomal DNA of human cells, are needed. Here, we developed a construct containing hygromycin resistance mutant gene fused to the EGFP gene as target for correction. HeLaS3 cells were transfected with the fusion gene and clones, which had integrated one or two copies of the mutated fusion gene, were isolated and expanded. These cells were transfected with a RDO with a mismatch at the position 336 of the bacterial hygromycin resistance gene. If the gene correction occurs, the expression of both hygromycin resistance and EGFP genes is recovered. The RFLP and FACS analysis demonstrated that hygromycin resistance phenotype was due to the correction of the mutation.
机译：嵌合RNA / DNA寡核苷酸（RDO）介导的真核细胞基因单碱基突变的基因校正仍是一个有争议的策略。为了更好地确定这种策略的潜力和适用性，需要新的系统来检测人细胞染色体DNA中RDO介导的基因校正。在这里，我们开发了一种构建体，该构建体包含与EGFP基因融合的潮霉素抗性突变基因作为校正目标。用融合基因转染HeLaS3细胞，分离并扩增整合了一或两个拷贝突变融合基因的克隆。这些细胞用在细菌潮霉素抗性基因的336位错配的RDO转染。如果发生基因校正，则潮霉素抗性和EGFP基因的表达都将恢复。 RFLP和FACS分析表明潮霉素抗性表型是由于突变的纠正所致。

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