MIP-MAP: High-Throughput Mapping of Caenorhabditis Elegans Temperature-Sensitive Mutants Via Molecular Inversion Probes

By Mok, Calvin A.; Au, Vinci et al. | Genetics, October 2017 | Go to article overview

MIP-MAP: High-Throughput Mapping of Caenorhabditis Elegans Temperature-Sensitive Mutants Via Molecular Inversion Probes


Mok, Calvin A., Au, Vinci, Thompson, Owen A., Edgley, Mark L., Gevirtzman, Louis, Yochem, John, Lowry, Joshua, Memar, Nadin, Wallenfang, Matthew R., Rasoloson, Dominique, Bowerman, Bruce, Schnabel, Ralf, Seydoux, Geraldine, Moerman, Donald G., Waterston, Robert H., Genetics


(ProQuest: ... denotes formulae omitted.)

The Million Mutation Project, a collection of genomic data for thousands of mutagenized Caenorhabditis elegáns strains (Thompson et al. 2013), has provided a dense library of mutant alleles with which to study gene function and has been widely used since its release. However, given that such strains generally contain only homozygous viable mutations, they largely exclude strongly deleterious alleles of essential genes. Such genes encompass a range of classes that include roles in cell division, development, and fertility; key components to all multicellular organisms. Temperature-sensitive (TS) lethal alleles can facilitate the genetic analysis of essential genes through the conditional modulation of their function without the complications that balancer chromosomes can introduce when present in nonconditional lethal strains (Golden et al. 2000; O'Rourke et al. 2011a; Lowry et al. 2015). There are several reported screens for TS lethal alleles in C. elegáns, but to date there are only a small portion of genes with TS alleles identified (Zonies et al. 2010; Ehmke et al. 2014; Lowry et al. 2015). Generating a comprehensive library of mutant strains with conditional lethal phenotypes has the potential to expand our knowledge of essential genes, their required levels of expression, the timing of their function(s), and the details of their protein and domain structure. Furthermore, such a catalog could have broad implications for elucidating the life cycle of C. elegans and other organisms. However, such an endeavor requires the mapping and characterization of mutant alleles in a systematic and high-throughput manner.

Chemical mutagenesis methods to generate TS alleles typically result in up to several hundred mutations per strain (Sarin et al. 2008; Flibotte et al. 2010; Thompson et al. 2013). Determining which of these molecular alterations is responsible for the TS mutant phenotype is especially challenging when dealing with partially penetrant or leaky alleles. Mutations that alter protein-coding sequence can be initially prioritized as candidates, but even after such filtering, > 1/5 of the collection's single-nucleotide variants (SNVs) remain. In contrast to identifying constitutive loss-of-function alleles-such as deletions, frameshifts, and nonsense mutations-there is, as yet, no universal predictive method for recognizing which coding or perhaps noncoding molecular change is most likely to give rise to a TS phenotype (Perry et al. 1994; Rogalski et al. 1995; Harfe et al. 1998; Poultney et al. 2011). When feasible, mutants are initially outcrossed or backcrossed to remove extraneous mutations originating from the mutagenesis process. Otherwise, these additional mutations may, to some degree, affect development or other essential pathways, thus obfuscating the process of allele characterization.

Genetic mapping is a general method for identifying the causative mutation in such strains. Mapping with classic visible genetic markers in C. elegans can be a laborious, iterative process. Single-nucleotide polymorphisms (SNPs) between strains, such as the N2 laboratory reference strain and the Hawaiian strain CB4856, act as molecular genetic markers that allow the parallel mapping of multiple sites across the genome in a single cross. The Snip-SNP assay is a popular method exploiting a subset of Hawaiian genome SNVs that alter recognition sites of the restriction enzyme DraI (Davis et al. 2005). However, the Hawaiian genome is proven to harbor various alleles (De Bono and Bargmann 1998; Seidel et al. 2008, 2011; Andersen et al. 2014) that can negatively alter the representation of segregant populations; CB4856 also has phenotypes of its own that may interfere with the scoring of some behavioral phenotypes (Wicks et al. 2001). Despite these issues, the research community continues to leverage CB4856 as a mapping strain to develop new methods of mapping complex mutations (Doitsidou et al. …

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