Up-regulation of luciferase gene expression with antisense oligonucleotides: Implications and applications in functional assay developments
Up-regulation of luciferase gene expression with antisense oligonucleotides: Implications and applications in functional assay developments
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DOI：
10.1021/bi980300h
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发表时间：
1998-05-05
期刊：
影响因子：
2.9
通讯作者：
Kole, R
中科院分区：
文献类型：
--
作者：
Kang, SH;Cho, MJ;Kole, R
用携带荧光素酶基因的重组质粒（pLuc/705）瞬时以及稳定转染HeLa Tet-Off细胞，所述荧光素酶基因被突变的人β-珠蛋白内含子2（IVS 2 -705）中断。内含子中的突变导致荧光素酶前体mRNA的异常剪接，阻止荧光素酶的翻译。然而，用靶向异常剪接位点的2 '-O-甲基寡核糖核苷酸处理细胞可诱导正确剪接，恢复荧光素酶活性。该作用具有序列特异性，取决于寡核苷酸的浓度，并且可以通过预处理细胞来调节。Luc/705，用四环素。因此，该细胞系尤其提供了一种优于基于蛋白质下调的其它方法的新型功能测定系统上级。特别地，该系统在评估反义寡核苷酸的细胞递送效率方面是理想的。
HeLa Tet-Off cells were transfected transiently as well as stably with a recombinant plasmid (pLuc/705) carrying the luciferase gene interrupted by a mutated human beta-globin intron 2 (IVS2-705). The mutation in the intron causes aberrant splicing of luciferase pre-mRNA, preventing translation of luciferase. However, treatment of the cells with a 2'-O-methyl-oligoribonucleotide targeted to the aberrant splice sites induces correct splicing, restoring luciferase activity, The effects are sequence-specific, depend on the concentration of the oligonucleotide, and can be modulated by the pretreatment of the cell line, Luc/705, with tetracycline. Thus, the cell line provides, among others, a novel functional assay system superior to other procedures that are based on protein down-regulation. In particular, the system would be ideal in assessing the cellular delivery efficiency of antisense oligonucleotides.