Efficient and cell specific knock-down of gene function in targeted C. elegans neurons
Introduction
Forward and reverse genetic analyses provide crucial contributions toward our understanding of how genes influence the behavior of multicellular organisms. However, many gaps still exist between our understanding of the functions of genes, which we describe in molecular and cellular terms, and our understanding of behavior, which we explain in terms of neural circuits and neurons. The nematode Caenorhabditis elegans has acquired a frontier role as a model in the analysis of behavior because its study promises to bridge some of these gaps, at least in a simple animal model (de Bono and Maricq, 2005). A critical step toward fulfilling this promise would be to find ways to determine the function that genes exert in each of the neurons/cells potentially responsible for a given behavioral trait (e.g. in each of the neurons composing a neural circuit). In C. elegans, beside traditional mosaic analysis, a commonly used approach to address this level of genetic analysis is the cell specific rescue of loss-of-function mutations (Fujiwara et al., 1999). Both approaches, although very useful, are generally laborious and time consuming and can only be applied to the study of genes for which true genetic mutants are available. The discovery of RNA interference (RNAi) has provided a powerful reverse genetics tool that has increased enormously the range of C. elegans genes whose function can be rapidly analysed (Fire et al., 1998, Kamath et al., 2003). However, even in C. elegans, there are several limitations to the use of RNAi to address the function of genes in specific cells, especially in neurons (Tavernarakis et al., 2000). First, direct delivery of dsRNA to worms, by injection, feeding or soaking (Ahringer, 2006) results in systemic RNAi which affects many or all the cells of the organism. It is thus not possible, with this approach, to dissect the role exerted by the gene of interest in specific cells or groups of cells and essential genes are obviously difficult to study because of lethality, sterility, etc. Second, some late acting genes and most genes expressed in neurons are largely refractory to RNAi (Tavernarakis et al., 2000, Ahringer, 2006). In C. elegans, transgene driven genetic interference has been described (Tavernarakis et al., 2000) as a possible way to overcome some of the limitations of classic RNAi obtained by direct delivery of dsRNA to the animals. The method of Tavernarakis et al. (2000) is based on the generation of inheritable and inducible genetic interference with hairpin dsRNA encoded by transgenes. However, expression of the hairpin dsRNA is driven by a strong and ubiquitously expressed heat-shock promoter and thus the method cannot be utilized for cell specific knock-downs (Tavernarakis et al., 2000, Johnson et al., 2005). Modifications of this approach, using cell specific promoters, have also been described (Tavernarakis et al., 2000, Timmons et al., 2003, Briese et al., 2006) but the efficiency of gene function knock-down in neurons and the cell autonomy of the effects obtained have either not been addressed or have produced conflicting results. In addition, problems related to the stability of plasmids, and possibly also of transgenes, carrying inverted repeat genes (Tavernarakis et al., 2000, Briese et al., 2006) have hindered so far a wider use of the inverted repeat approach to study gene function in C. elegans. Inducible genetic interference has also been obtained with sense and antisense RNAs transcribed as separate molecules and not as a hairpin from inverted repeat sequences (Gupta et al., 2003). However, also in this case, a ubiquitously expressed heat-shock promoter was used and thus the knock-down of the gene function was not targeted to specific cells. In addition, the reduction of gene function in neurons was not analysed (Gupta et al., 2003). Here we describe a simple transgene driven approach and show that it produces an efficient and heritable knock-down of gene function in chosen neurons of C. elegans.
Section snippets
Strains
Wild-type animals were C. elegans variety Bristol, strain N2. Alleles used in this work included osm-10(n1602)III, osm-6(p811)V and were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). A strain carrying an integrated transgene in which the gpa-15 promoter is fused to the gfp gene Is[pgpa-15::gfp] (Jansen et al., 1999), was kindly provided by G. Jansen (Rotterdam, The Netherlands). The name of the strains and the genotype
Construction of transgenes for cell specific knock-down
We tested whether a heritable reduction of gene function in chosen C. elegans neurons could be efficiently and simply achieved with transgenes from which sense and antisense RNAs, corresponding to the gene of interest, are transcribed as separate molecules by cell specific promoters. If this were the case, a simpler and faster strategy for the construction of the appropriate transgenes could be followed. For each interference we fused, by co-amplification, an exon rich region of the gene under
Discussion
The goal of the method presented here is to determine the function of a gene in specific cells, including neurons. The procedure results in the efficient knock-down of gene function in chosen C. elegans neurons. The effect observed is cell autonomous and heritable. The method can also be usefully applied to ubiquitously expressed essential genes and possibly to other cell types. We do not know whether the reduction of gene function obtained is due to post-transcriptional or transcriptional
Acknowledgements
We wish to thank S. Arbucci and A. Sollo for skilled technical assistance and F. Cernilogar, U. Di Porzio, F. Graziani, A. La Volpe for suggestions and for reading the manuscript. This work was supported in part by Telethon grant No. GGP030288 and by FIRB-Neuroscience grant RBNE01WY7P.
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These two authors contributed equally to the work.