Automated screening for mutants affecting dopaminergic neuron specification in C. elegans

We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

[1]  R. Pulak,et al.  Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system. , 2006, Methods in molecular biology.

[2]  C. Guenther,et al.  Asymmetric distribution of the C. elegans HAM-1 protein in neuroblasts enables daughter cells to adopt distinct fates. , 1996, Development.

[3]  M. Götz,et al.  Neuronal fate determinants of adult olfactory bulb neurogenesis , 2005, Nature Neuroscience.

[4]  S. Brenner The genetics of Caenorhabditis elegans. , 1974, Genetics.

[5]  I. Pe’er,et al.  Caenorhabditis elegans mutant allele identification by whole-genome sequencing , 2008, Nature Methods.

[6]  Michael R Koelle,et al.  Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans , 2004, Nature Neuroscience.

[7]  David H. Hall,et al.  Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Matthew M. Crane,et al.  Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans , 2008, Nature Methods.

[9]  A. Abeliovich,et al.  Midbrain dopamine neuron differentiation: factors and fates. , 2007, Developmental biology.

[10]  J. Bessereau,et al.  Mos1-mediated insertional mutagenesis in Caenorhabditis elegans , 2007, Nature Protocols.

[11]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[12]  R. Blakely,et al.  A genetic screen in Caenorhabditis elegans for dopamine neuron insensitivity to 6‐hydroxydopamine identifies dopamine transporter mutants impacting transporter biosynthesis and trafficking , 2005, Journal of neurochemistry.

[13]  Scott W. Emmons,et al.  A transcription factor controlling development of peripheral sense organs in C. elegans , 1995, Nature.

[14]  Erik M. Jorgensen,et al.  The art and design of genetic screens: Caenorhabditis elegans , 2002, Nature Reviews Genetics.

[15]  Patrick W. Hullett,et al.  BMC Genomics BioMed Central Methodology article Rapid single nucleotide polymorphism mapping in C. elegans , 2005 .