Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences.
Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences.
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DOI：
10.1093/abbs/gmp024
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发表时间：
2009-05
影响因子：
3.7
通讯作者：
Zhifeng Zeng;Hongjian Li;Yue-qin Li;Yanwei Cui;Qi Zhou;Yi Zou;Guang Yang;Tian-hong Zhou
中科院分区：
文献类型：
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作者：
Zhifeng Zeng;Hongjian Li;Yue-qin Li;Yanwei Cui;Qi Zhou;Yi Zou;Guang Yang;Tian-hong Zhou
为了研究12个核苷酸的miniEGSs是否能有效沉默人巨细胞病毒（HCMV）UL 49基因的表达，构建了稳定表达UL 49基因的HeLa细胞系，并在稳定细胞系中检测了推定的miniEGSs（UL 49-miniEGSs）。定量RT-PCR和蛋白质印迹结果显示，在用UL 49-miniEGSs转染的HeLa细胞中，UL 49表达水平降低了67%。与模拟和对照miniEGSs（TK-miniEGSs）的1%和7%相比，差异显著。为了进一步证实UL 49-miniEGSs与人RNase P的基因沉默，构建了UL 49-miniEGSs的突变体，并进行了修饰的5 'RACE。结果表明，UL 49-miniEGSs对UL 49基因表达的抑制作用是RNase P依赖性的，并且RNase P对UL 49 mRNA的切割具有位点特异性。结果表明，能够有效沉默基因表达的基于DNA的miniEGSs长度仅为12 nt。这明显低于迄今为止已知的任何其他基于阿糖胞苷的基因失活方法。MiniEGSs可能代表用于抑制病毒基因和其他人类疾病相关基因表达的新型基因靶向剂。
To investigate whether a 12 nucleotide DNA-based miniEGSs can silence the expression of human cytomegalovirus (HCMV) UL49 gene efficiently, A HeLa cell line stably expressing UL49 gene was constructed and the putative miniEGSs (UL49-miniEGSs) were assayed in the stable cell line. Quantitative RT-PCR and western blot results showed a reduction of 67% in UL49 expression level in HeLa cells that were transfected with UL49-miniEGSs. It was significantly different from that of mock and control miniEGSs (TK-miniEGSs) which were 1% and 7%, respectively. To further confirm the gene silence directed by UL49-miniEGSs with human RNase P, a mutant of UL49-miniEGSs was constructed and a modified 5'RACE was carried out. Data showed that the inhibition of UL49 gene expression directed by UL49-miniEGSs was RNase P-dependent and the cleavage of UL49 mRNA by RNase P was site specific. As a result, the length of DNA-based miniEGSs that could silence gene expression efficiently was only 12 nt. That is significantly less than any other oligonucleotide-based method of gene inactivation known so far. MiniEGSs may represent novel gene-targeting agents for the inhibition of viral genes and other human disease related gene expression.