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A multiply fluorescent strain for outcrossing Million Mutation Project alleles and linkage mapping

Jonathan Hodgkin
1Department of Biochemistry, University of Oxford, Oxford UK

Correspondence to: Jonathan Hodgkin (jonathan.hodgkin@bioch.ox.ac.uk)

The Million Mutation Project (Thompson et al., 2013) has provided a wonderful resource to the C. elegans community. However, the 2007 strains generated by this project each carry about 400 mutations, on average, so it is essential to remove as much as possible of the irrelevant and potentially confusing background of linked and unlinked mutations when investigating a MMP mutation in one particular gene. Repeated outcrossing with wild-type strains can eventually remove most of the unlinked background, but this is a random process that takes time and is not guaranteed to achieve completion. Complete replacement of LGX can be readily achieved by transmission through a male carrying a wild-type X chromosome, but explicitly replacing autosomes is harder. Introducing dominantly marked chromosomes, and then replacing them with wild-type chromosomes, allows easy and explicit removal of all extraneous mutations in the central region of each autosome, where most genes are located. For this purpose, a strain has been constructed that carries a set of integrated fluorescent transgene insertions, one for each of LGI, II, IV and V, plus a visible marker on LGIII. All of the fluorescent markers are dominant, homozygous viable, and located in the autosomal cluster regions. All can be easily and independently scored using a dissecting microscope equipped with epifluorescence. The strain is also generally useful for assigning linkage to unmapped mutations. The genotype of this strain (CB7272) is: ccIs4251 I; mIs12 II; dpy-17(e164) III; frIs7 IV; uIs69 V Its main phenotypes are: green fluorescence in body wall muscle nuclei (ccIs4251 I), green fluorescence in pharyngeal muscle (mIs12 II), Dumpy (dpy-17 III), red fluorescence in epidermis (frIs7 IV), red fluorescence in pharyngeal muscle (uIs69 V). Surface infection, injury, or osmotic stress might induce green fluorescence in epidermis (due to frIs7), and the strain might exhibit hypersensitive neuronal RNAi by feeding (due to uIs69), but these additional phenotypes can usually be ignored. The insertion ccIs4251 is associated with a recessive male mating defect, for which reason it is convenient to mate CB7272 hermaphrodites with wild-type males, before crossing the resulting multiply heterozygous males with the strain of interest. CB7272 was constructed using transgenic lines available from the Caenorhabditis Genetics Center (CGC). Undoubtedly, better versions of this kind of strain could be constructed, using integrated transgenes that have not yet been deposited at the CGC. An improved strain might include additional distinguishable fluorescent markers for LGIII and LGX, would avoid using ccIs4251 (in view of its low male mating efficiency) and would contain a him-5 or him-8 mutation to generate homozygous males. Many thanks are due to the Fire, Riddle, Ewbank and Chalfie labs for creating and making available the transgene insertions used for construction. The strain CB7272 has been deposited at the CGC and can be obtained from there.

References

Thompson O, Edgley M, Strasbourger P, Flibotte S, Ewing B, Adair R, Au V, Chaudhry I, Fernando L, Hutter H, et al. (2013). The Million Mutation Project: a new approach to genetics in Caenorhabditis elegans. Genome Res. 23, 1749-1762.  PubMed

Endogenous locus modification by genome engineering: proposed nomenclature

Tim Schedl1 and Jonathan Hodgkin2
1Department of Genetics, Washington University School of Medicine, St Louis MO, 1Department of Biochemistry, University of Oxford, Oxford, UK

Correspondence to: Tim Schedl,Jonathan Hodgkin (ts@genetics.wustl.edu)

1Present and 2Past WormBase Gene Name Curators.

New methods for genome engineering (TALENs, CRISPR-Cas9, etc.) are increasingly being applied to C. elegans. These entail some additional recommendations to the standard Genetic Nomenclature Guidelines (http://www.wormbase.org/about/userguide/nomenclature), as described below. The aim is to provide compact and unambiguous ways of describing and referring to engineered changes to endogenous loci, as distinct from transgenic constructs that are inserted elsewhere in the genome.

Each engineered modification to an endogenous locus (point mutations, deletions, insertions or combinations thereof) should receive a unique allele designation, using the standard allele designation of the originating laboratory. For example: bus-50(e5000).

Optional brackets can be employed to provide additional information.

Example: bus-50(e5000[T110E]) (an engineered missense mutation).

An engineered fusion of GFP to the C-terminus of BUS-50 would be:

bus-50(e5001[bus-50::gfp]).

As a shorter and more convenient form, and where unambiguous, this could be referred to as:

bus-50::gfp. Such abbreviations should be clearly defined where first used in a paper.

An engineered insertion of GFP plus the unc-119(+) selectable marker, flanked by loxP sites, would be:

bus-50(e5002[bus-50::gfp + loxP unc-119(+) loxP]).

Each additional engineering of the endogenous locus requires a new allele number. In the example of bus-50(e5002), following Cre-mediated recombinase removal of unc-119(+) so that a single loxP site remains, the new genotype would be bus-50(e5003[bus-50::gfp +loxP]) or bus-50(e5003) for short.

Engineered insertions in apparent intergenic regions are given standard Is insertion names, for example eIs2002. Optional descriptors can include the nature of the insertion, e.g., [unc-119::gfp] and the position in the genome, e.g., [III:2992500], to give eIs2002[unc-119::gfp] or eIs2002[unc-119::gfp, III:2992500].

Engineered changes to existing Is (or Si) insertions should receive new Is numbers using originating lab’s prefix. The original Is insertion can be indicated in brackets with a preceding asterisk (*), in order to allow searches for all derivatives from a given insertion.

For example, an engineered change from GFP to mCherry in eIs2002 might be named as ozIs909, or ozIs909[unc-119::mCherry *eIs2002].

Notable recent papers in genetics and genomics

Jonathan Hodgkin, editor of the Genetics and genomics section of WormBook
1Department of Biochemistry, University of Oxford, Oxford UK

Correspondence to: Jonathan Hodgkin (jonathan.hodgkin@bioch.ox.ac.uk)

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Chang HC, Paek J, and Kim DH. (2011). Natural polymorphisms in C. elegans HECW-1 E3 ligase affect pathogen avoidance behaviour. Nature 480, 525-529.   PMID: 22089131

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Melo JA, and Ruvkun G. (2012). Inactivation of conserved C. elegans genes engages             pathogen-and xenobiotic-associated defenses. Cell 149, 452-466.  PMID: 22500807

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