Published online 28 April 2005
Methods Online |
Suppression of vascular endothelial growth factor expression at the transcriptional and post-transcriptional levels
ToolGen, Inc. 461-6, Jeonmin-dong, Yuseong-gu, Daejeon 305-390, South Korea
*To whom correspondence should be addressed. Tel: +82 42 863 8166; Fax: +82 42 863 3840; Email: jsk{at}toolgen.com
Received January 10, 2005. Revised March 30, 2005. Accepted March 30, 2005.
Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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