Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells

BMC Biotechnology

Research Article

Kousei Noguchi¹  Hiroshi Takaku¹  Yoshio Ishitu¹ 

[1] Department of Life and Environmental Science, Chiba Institute of Technology, 2-17-1 Tsudanuma, 275-0016, Narashino, Chiba, Japan;

关键词: Theophylline;    293T Cell;    Renilla Luciferase;    Chaperone Protein;    Antisense Strand;   

DOI  :  10.1186/1472-6750-11-79

 received in 2010-04-16, accepted in 2011-07-25,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundThe group I intron, a ribozyme that catalyzes its own splicing reactions in the absence of proteins in vitro, is a potential target for rational engineering and attracted our interest due to its potential utility in gene repair using trans-splicing. However, the ribozyme activity of a group I intron appears to be facilitated by RNA chaperones in vivo; therefore, the efficiency of self-splicing could be dependent on the structure around the insert site or the length of the sequence to be inserted. To better understand how ribozyme activity could be modulated in cultured mammalian cells, a group I intron was inserted into a short hairpin RNA (shRNA), and silencing of a reporter gene by the shRNA was estimated to reflect self-splicing activity in vivo. In addition, we appended a theophylline-binding aptamer to the ribozyme to investigate any potential effects caused by a trans-effector.ResultsshRNA-expression vectors in which the loop region of the shRNA was interrupted by an intron were constructed to target firefly luciferase mRNA. There was no remarkable toxicity of the shRNA-expression vectors in Cos cells, and the decrease in luciferase activity was measured as an index of the ribozyme splicing activity. In contrast, the expression of the shRNA through intron splicing was completely abolished in 293T cells, although the silencing induced by the shRNA-expressing vector alone was no different from that in the Cos cells. The splicing efficiency of the aptamer-appended intron also had implications for the potential of trans-factors to differentially promote self-splicing among cultured mammalian cells.ConclusionsSilencing by shRNAs interrupted by a group I intron could be used to monitor self-splicing activity in cultured mammalian cells, and the efficiency of self-splicing appears to be affected by cell-type specific factors, demonstrating the potential effectiveness of a trans-effector.

【 授权许可】

Unknown   
© Noguchi et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.