Email updates

Keep up to date with the latest news and content from BMC Molecular Biology and BioMed Central.

Open Access Highly Accessed Research article

Cell- and region-specific miR30-based gene knock-down with temporal control in the rat brain

Beihui Liu1*, Haibo Xu12, Julian FR Paton1 and Sergey Kasparov1*

Author Affiliations

1 Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, UK

2 Department of Pharmacology, State Key Laboratory for Research and Development of Chinese Materia Medica, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, P.R. China

For all author emails, please log on.

BMC Molecular Biology 2010, 11:93  doi:10.1186/1471-2199-11-93

Published: 6 December 2010

Abstract

Background

RNA interference (RNAi) emerges as a powerful tool to induce loss-of-function phenotypes. In the context of the brain, gene manipulation is best targeted to specific subsets of cells in order to achieve a physiologically relevant outcome. Polymerase II-based viral expression systems can be used to cell-specifically express constructs incorporating flanking and loop sequences from endogenous microRNA (miRNA), which directs the designed hairpins into the endogenous gene silencing machinery. While many studies have documented non-cell-selective gene knock-down in the brain, it has not been tested whether different cell types or different areas of the central nervous system (CNS) are equally amenable to this approach. We have evaluated this issue using a tetracycline (Tet)-controllable and cell-specific miRNA 30 (miR30)-based short hairpin (shRNA) interference system.

Results

To achieve targeted expression two cell type-specific promoters were used; the enhanced compact glial fibrillary acidic protein (GfaABC1D) promoter and the enhanced human synapsin-1 (SYN) promoter. Powerful luciferase (Luc) and the neuronal isoform of nitric oxide synthase (nNOS) gene knock-down were achieved both in vitro and in vivo. Administration of doxycycline (Dox) abrogated gene silencing. However, the efficacy of gene knock-down in both neurones and astrocytes in the hippocampus (HIP) was lower than that in the dorsal vagal complex of the brainstem (DVC). This was not due to regional differences in the expression of the the key enzymes involved in miRNA processing.

Conclusions

The results from the presented experiments demonstrated that selective gene knock-down in subsets of brain cells is achievable. However, there are some presently unknown regional factors which affect either the processing of miRNA-based cassettes or their potency for gene silencing.