Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences.
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.
Zeng
Zhifeng
Z
Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China.
Li
Hongjian
H
Li
Yueqing
Y
Cui
Yanwei
Y
Zhou
Qi
Q
Zou
Yi
Y
Yang
Guang
G
Zhou
Tianhong
T
D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043262:Ribonuclease P / Q000378:metabolism*; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D015678:Viral Structural Proteins; D043262:Ribonuclease P