We have been much involved in developing, improving and customizing methodologies to further exploit the specific advantages of C.elegans as a genetic model system.
We have continuously worked on improving transgenic reporter tools. Apart from describing practical considerations[1, 2], we have developed a robust and fast pipeline for generating fosmid-based reporters through a bacterial recombineering strategy.
rab-3::yfp labeling all neurons
(b) Automated Screening
To facilitate the ultimate goal of genetic analysis – saturation of a mutationally probed biological process – we have automated reporter-based C.elegans mutant selection. We have used this technology to elucidate the gene regulatory logic of dopamine neuron specification[4, 5], and are currently applying this technology to several other mutant screens.
Copas biosort (aka “wormsorter”)
(c) Whole Genome Sequencing
We have pioneered the use of whole genome sequencing (WGS) to pinpoint mutagen-induced molecular lesions, thereby shortcutting time-consuming positional cloning[6, 7]. We have compared sequencing platforms, developed customized software to make this approach widely accessible[9, 10] and developed a combined SNP-mapping/WGS strategy that we think represents the ultimate method for mutant identification in C.elegans. We have employed WGS to identify molecular lesions in many different strains[7, 12-15] and discovered unanticipated features of mutagenized genomes.
Genetics cover (June 2010 vol. 185 no. 2). Image designed by Aleksandra Rogula
1. Etchberger, J.F. and O. Hobert, Vector-free DNA constructs improve transgene expression in C. elegans. Nat Methods, 2008. 5(1): p. 3.
2. Hobert, O., PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques, 2002. 32(4): p. 728-30.
3. Tursun, B., et al., A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans. PLoS ONE, 2009. 4(3): p. e4625.
4. Flames, N. and O. Hobert, Gene regulatory logic of dopamine neuron differentiation. Nature, 2009. 458(7240): p. 885-9.
5. Doitsidou, M., et al., Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans. Nat Methods, 2008. 5(10): p. 869-72.
6. Hobert, O., The impact of whole genome sequencing on model system genetics: get ready for the ride. Genetics, 2010. 184(2): p. 317-9.
7. Sarin, S., et al., Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nat Methods, 2008. 5(10): p. 865-7.
8. Shen, Y., et al., Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing. PLoS ONE, 2008. 3(12): p. e4012.
9. Bigelow, H., et al., MAQGene: software to facilitate C. elegans mutant genome sequence analysis. Nat Methods, 2009. 6(8): p. 549.
10. Minevich, G., et al., CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences. Genetics, 2012. 192(4): p. 1249-69.
11. Doitsidou, M., et al., C. elegans Mutant Identification with a One-Step Whole-Genome-Sequencing and SNP Mapping Strategy. PLoS ONE, 2010. 5(11): p. e15435.
12. Zhang, F., M.M. O’Meara, and O. Hobert, A left/right asymmetric neuronal differentiation program is controlled by the Caenorhabditis elegans lsy-27 zinc-finger transcription factor. Genetics, 2011. 188(3): p. 753-9.
13. Sarin, S., et al., Analysis of multiple ethyl methanesulfonate-mutagenized caenorhabditis elegans strains by whole-genome sequencing. Genetics, 2010. 185(2): p. 417-30.
14. Flowers, E.B., et al., The Groucho ortholog UNC-37interacts with the short Groucho-like protein LSY-22 to control developmental decisions in C. elegans. Development, 2010. 137: p. 1799-1805.
15. Poole, R.J., et al., A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans. PLoS Genet, 2011. 7(6): p. e1002109.