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Acta Biochimica et Biophysica Sinica Advance Access published online on March 31, 2009
Acta Biochimica et Biophysica Sinica, doi:10.1093/abbs/gmp024
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences

Zhifeng Zeng, Hongjian Li, Yueqing Li, Yanwei Cui, Qi Zhou, Yi Zou, Guang Yang and Tianhong Zhou*

Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou 510632, People's Republic of China

* Corresponding author. Tel: +86-20-85-22-3087; Fax: +86-20-85-22-3087; E-mail: tzth{at}jnu.edu.cn


  Abstract

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.

Keywords    DNA-based miniEGSs; RNase P; gene silence; human cytomegalovirus; antivirus

Received: December 1, 2008; Accepted: March 10, 2009
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