Specific interference with gene expression and gene function mediated by long dsRNA in neural cells
Introduction
Specific gene silencing mediated by double-stranded RNA (dsRNA), or RNA interference (RNAi), has been widely used to study gene function in diverse invertebrate systems, such as C. elegans and Drosophila (Fire et al., 1998, Kennerdell and Carthew, 1998, Hammond et al., 2000, Sharp and Zamore, 2000). The identification of a small RNA species of ∼25 bp nucleotides in plants undergoing post-transcriptional silencing (PTGS) provided a key insight into the understanding of the mechanistic basis of RNAi (Hamilton and Baulcombe, 1999). In cell-free systems with extracts of Drosophila embryos and Schneider cells, dsRNA is processed into 21–25 nt small inhibitory RNA (siRNA), which guides the degradation of target mRNA at 21–23 nt intervals (Tuschl et al., 1999, Zamore et al., 2000).
Unlike invertebrate cells, most mammalian cells respond to dsRNAs longer than 30 bp by inducing interferon α and β expression and activating dsRNA-dependent protein kinase (PKR) and 2′, 5′ oligoadenylate synthetase, leading to a general inhibition of protein synthesis and non-specific degradation of mRNA by RNase L (Stark et al., 1998). However, potent and specific dsRNA-mediated inhibition of gene expression was observed in mouse embryos microinjected with long dsRNA, suggesting the existence of the RNAi machinery in mammalian cells (Wianny and Zernicka-Goetz, 2000). Use of RNAi in mammalian cells was further validated and greatly expanded by Tuschl and colleagues, in which siRNAs were used to induce gene-specific silencing in a variety of mammalian cell lines (Elbashir et al., 2001). However, potent and specific RNAi-mediated silencing was not achieved with long dsRNA in commonly used mammalian cells, including Hela, HEK293, CHO, NIH3T3 cells, due to non-specific inhibition of gene expression (Ui-Tei et al., 2000, Elbashir et al., 2001). Recent studies reported that long dsRNA-induced efficient RNAi in non-differentiated mouse embryonic teratocarcinoma cell lines which are deficient in some of the dsRNA- and IFN-activated enzymes (Billy et al., 2001, Yang et al., 2001, Paddison et al., 2002).
In this report we show that dsRNAs with an average size of 500–700 base pairs are able to mediate gene-specific silencing of an integrated transgene and endogenous genes in partially and fully differentiated mouse neuroblastoma AGYNB010 cells, derived from N2a cells. Using AGYNB010 cells that stably express green fluorescent protein (GFP), we show that the expression level of GFP is significantly attenuated by dsRNA derived from the GFP ORF, but not by dsRNA derived from unrelated genes. In addition, we have shown efficient silencing of several endogenous genes involved in apoptosis, housekeeping, and signal transduction. RNAi-mediated gene silencing was then used as an efficient knockdown tool to confirm the role of an endogenous gene, PARP, in oxygen–glucose deprivation (OGD)-induced neuronal damage (Eliasson et al., 1997). Transfection of dsRNA derived from either the N-terminal or C-terminal part of PARP ORF strongly inhibited expression of PARP. Moreover, inhibition of PARP expression induced cellular resistance to OGD, which is consistent with the role of the PARP protein in ischemia-induced brain damage. Our results indicate that RNAi-mediated gene silencing is a useful tool to elucidate gene function in these neural cells.
Section snippets
Double-stranded RNA
Sequence-specific primers were used to generate partial clones to be inserted into PCR4TOPO, which served as templates for in vitro transcription to generate sense and antisense transcripts using T3 and T7 polymerases (Ambion). An equal molar ratio of sense and antisense transcripts were heated at 60 °C for 30 min and cooled to room temperature for more than 15 min for annealing. The dsRNA preparation was treated with RNase T1 to eliminate any remaining single-stranded RNA. The quality of the
Inhibition of GFP expression in AGYNB010 cells stably expressing GFP
To demonstrate whether long dsRNA mediates gene-specific silencing in mouse neuroblastoma AGYNB010 cells, we first chose the transgene GFP as a reporter gene. Unlike other studies in which the expression plasmid coding for the reporter gene was cotransfected with dsRNA in a transient manner, we generated an NB010-GFP cell line that expresses GFP protein stably to mimic the expression of an endogenous gene. dsRNA corresponding to the full-length (dsGFP) or to the C-terminal part of the GFP
Discussion
In this report, we show for the first time long dsRNA-induced gene-specific silencing in mouse neuroblastoma AGYNB010 cells under fully or partially differentiated conditions. We demonstrate dsRNA-induced gene-specific silencing of an integrated transgene as well as of a variety of endogenous genes involved in apoptosis, signaling, and housekeeping functions. Control dsRNA did not induce non-specific inhibition of the cognate gene expression. Expression of unrelated housekeeping genes, MAP2 and
Acknowledgements
The authors would like to thank Drs Roman Urfer and Mehrdad Shamloo for their helpful comments.
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