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Brief report on the use of the phiC31 integrase for the transgenic array engineering

Wadim Kapulkin1,*, Andrei Pozniakovsky1
1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
* present affiliation: Veterinary Consultancy, Conrada 16/65, 01-922 Warsaw, Poland
Correspondence to: Wadim Kapulkin (wadim_kapulkin@yahoo.co.uk)
In systems other than worms (i.e. and also vertebrate cells) Streptomyces temperate phage phiC31 integrase was reported as an efficient mean of inserting the transgenes into specific sites of the genome. In the summer of 2013 we have made an experimental attempt to evaluate if phiC31 integrase would be of possible use in C. elegans. For this purpose we implemented the unc-119(ed3) mutant rescue (1), by linking of two split segments of the unc-119 rescuing cassette. Split segments of unc-119 are two separate plasmids providing respectively: unc-119 promoter and N-terminal UNC-119 CDS and C-terminal UNC-119 followed by unc-119 3′ utr (split at the third intron and flanked with the appropriate phiC31 recognition sites). In addition the third construct was used, intended to provide the phiC31 integrase into the germline cells (essentially based on pJL43.1 – p-glh-2::MosTase::glh-2-utr, where the Mos1 transposase (2, 3) was replaced by codon optimized phiC31 integrase) to facilitate the transgene engineering.

Using the above constructs, we obtained the following evidence for the phiC31 acting efficiently on the transgenic extrachromosomal arrays. We assumed here, the two separate plasmids containing split of two segments of unc-119 gene will rescue the ed3 allele in DP38 derived strain, only if properly re-assembled into functional unc-119 gene. To test for the above assumption the above plasmids, where co-injected into unc-119(-) hermaphrodites carrying homozygous ed3 allele. In the first experiment, two of the above plasmids containing split of two segments of the unc-119 gene, were injected (along with fluorescent markers*) into gonads of the DP38 hermaphrodites. Based on the segregation of the fluorescent markers in the transgenic progeny in this experiment, we concluded that while the injected split unc-119 segments do efficiently form the heritable transgenic array, however fail to rescue the uncoordinated phenotype (observed at least four 4 independent array forming events in 30 injected hermaphrodites).

We contrast the above observation, with the results of the second experiment, where the above plasmids where co-injected with the phiC31 integrase providing plasmid (again along with fluorescent markers) into gonads of DP38 derived hermaphrodites. We observed at least 4 independent events (in 28 injected hermaphrodites) of formation of the heritable arrays resulting in non-uncoordinated movement in unc-119(-) animals, when the split segments of unc-119 were provided with the third plasmid encoding for phiC31 integrase. Consistently the above four of the unc-119 rescued lines, were proven resistant to G418 indicating for the transmission of the selectable marker on the extrachromosomal array and the restored unc-119 function, depending strictly on the germinal provision of the phiC31 expressing plasmid. Collectively, we think we do have  enough evidence, to conclude the phiC31 is working on the transgenic arrays formed of the split unc-119 segments. We think this demonstration could be possibly useful in for example array-array transgenic engineering technology i.e. phiC31 targeted  linking of the two separate extrachromosomal arrays.

* those lines where propagated under G418 selection, as the intact NeoR gene was included into one of the constructs, providing additional line of the confidence over the selection based on fluorescent markers when maintaining the transgenic arrays in the (slow growing) non-rescued uncoordinated unc-119(-) line.

Acknowledgments: We are grateful to Mihail Sarov (www.mpi-cbg.de/services-facilities/core-facilities/genome-engineering-facility/), for providing the p-glh-2 driven phiC31 integrase construct and the friendly and supportive laboratory environment.

References

Maduro M, Pilgrim D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics. 1995 Nov;141(3):977-88.   PubMed

Bessereau JL, Wright A, Williams DC, Schuske K, Davis MW, Jorgensen EM. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature. 2001 Sep 6;413(6851):70-4.
  PubMed

Frøkjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM. Single-copy insertion of transgenes in Caenorhabditis elegans
Nat Genet. 2008 Nov;40(11):1375-83. doi: 10.1038/ng.248.
  PubMed

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Myths and misconceptions about C. elegans ecology and evolution

Asher D. Cutter1, Marie-Anne Félix2 and Mark Blaxter3
1Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada
2 Institute of Biology of Ecole Normale Supérieure, Paris, France
3 Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom
Correspondence to: Asher D. Cutter (asher.cutter@utoronto.ca)
C. elegans biologists show an admirable desire to understand all facets of the worm’s biology, often wanting to relate their work to the context of C. elegans as a “real organism” that lives out in the wild world. However, some early perceptions about C. elegans biology are now known to be incorrect. Here we highlight four common misconceptions to, hopefully, eliminate the perpetuation of these myths.

C. elegans is not a soil nematode: it is found in niches rich in decomposing organic matter. Despite old statements of C. elegans living in soil, no species of Caenorhabditis is found commonly in dirt – although Caenorhabditis are terrestrial when compared to marine nematodes. To understand why, it helps to think like a hungry worm. Like all species in the genus, C. elegans eats bacteria and some other microbes, and consequently C. elegans (and related species) occur in the wild with greatest prevalence in niches rich in decomposing organic matter that have abundant bacterial growth, such as rotting fruits, rotting flowers, and rotting plant stems. Compost bins are an analogous, human-constructed niche.

C. elegans and C. briggsae did not diverge 100 million years ago. The rapid generation time and somewhat higher mutation rate per generation of Caenorhabditis mean that sequence divergence accumulates faster per year than in many other organisms. Consequently, date estimates based on ‘universal’ molecular clock calibrations drastically overestimate divergence times of C. elegans with other organisms. The lack of a fossil record, the uncertainty in average generation turnover per year, and the extensive sequence divergence between C. elegans and its relatives all make it difficult to estimate accurately divergence times. However, best current estimates imply that C. elegans and C. briggsae diverged less than 30 million years ago (see Cutter 2015). Current published dates for the divergence of C. elegans and Pristionchus pacificus have no analytic support (Blaxter 2009).

N2 is not a “wild” genetic background, but is a strain adapted to the laboratory. Like all organisms reared for many generations in the lab, C. elegans N2 has adapted to the bench, perhaps aided by unintentional artificial selection. The genetic causes of several major lab adaptations are known in detail (npr-1, glb-5, nath-10), which exact numerous pleiotropic effects (Sterken et al. 2015). The domesticated strain N2 may thus poorly predict the biology of wild strains for many traits, and epistasis may modify the phenotypic outcome of many mutations.

Selfing hermaphroditism (protandrous dioecy) is an unusual mode of reproduction in nematodes. Despite the famous experimental power of self-fertilization in C. elegans, its ancestors and 95% of the 52 known Caenorhabditis species reproduce by obligatory mating between males and females. Although a diversity of C. elegans’ features reflect adaptation to a selfing lifestyle with few males (e.g. hermaphrodite resistance to mating, loss of sex-biased genes, small sperm), most traits shared with other Caenorhabditis require explanation in the evolutionary context of outbreeding ancestral species.

References

Blaxter M (2009). Nematodes (Nematoda). In: The Timetree of Life, SB Hedges and S Kumar, eds. Oxford Univ. Press. Pp. 247-250. (http://timetree.org/book)

Cutter AD (2015). Bioessays. 37: 983-995.  PubMed

Sterken MG, et al. (2015). Trends Genet 31: 224-231.  PubMed

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Dorsal Dauers and Directionally Dysfunctional dex-1 Dumpies

Caroline D. Beshers1,2, Kristen M. Flatt1,3 and Nathan E. Schroeder1,3
1 Crop Sciences Department, University of Illinois Urbana-Champaign
2 Neuroscience Department, Oberlin College, Ohio
3 Neuroscience Program, University of Illinois Urbana-Champaign

Correspondence to: Caroline Beshers (caroline.beshers13@gmail.com)
The dauer life stage in wild-type C. elegans exhibits morphological and behavioral characteristics that differentiate it from non-dauers (Hu, 2007). In addition to the commonly observed radial shrinkage, quiescence and lack of pharyngeal pumping, we noted a tendency of wild-type dauers to lie dorsoventrally when mounted on agarose slides, as opposed to laterally like adults. To quantify this observation, we mounted wild-type dauers and L3s on increasing concentrations of agarose with 0.1 M levamisole. We found that 75-90% of wild-type dauers mounted with levamisole lie dorsoventrally regardless of agarose concentration (Fig. 1). Very few (2.5-5%) non-dauer animals were positioned dorsoventrally. There was no significant difference in positioning between dauers that still retained their L2d cuticle and those that were unsheathed.  We then utilized a non-anesthetic method of immobilization by mounting dauers on 10% agarose with 10 micron polystyrene microbeads (Kim et al., 2013). Interestingly, only 15% of wild type dauers lie dorsoventrally when mounted using the microbeads. We hypothesized that the radial shrinkage and the presence of lateral alae in wild-type dauer cuticles may cause anesthetized dauers to roll to a dorsoventral position, but that “conscious” dauers immobilized with microbeads would remain lateral, despite the presence of alae. To test this, we examined dex-1 mutant dauers. We recently found that dex-1(ns42) dauers are defective for dauer alae formation and radial constriction leading to a dumpy dauer phenotype. We found that dex-1 dauers are significantly more likely to lay laterally than wild-type dauers (p=0.0013) (Fig. 1) suggesting that dauer alae or overall body dimensions regulate the anesthetized “sleeping” position.

Figures



Figure 1: This chart illustrates the difference in percentage of N2 dauers, N2 L3s and dex-1 dauers lying dorsoventrally across slide methods with increasing concentrations of agarose using levamisole (lev) and 10% agarose using polystyrene microbeads (beads). The N2 dauers consistently (75-90%) lay dorsoventrally, while 2-5% of non dauer animals lay dorsoventrally. N2 dauers lay dorsoventrally significantly more than dex-1 dauers (p=.0013).


Figure 2:

References

Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook  PubMed

Kim E, Sun L, Gabel CV, Fang-Yen C (2013) Long-Term Imaging of Caenorhabditis elegans Using Nanoparticle-Mediated Immobilization. PLoS ONE 8(1): e53419.   PubMed

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NEW LAB ANNOUNCEMENT: Jordan D. Ward

Jordan D. Ward
Department of Molecular, Cell, and Developmental Biology
University of California, Santa Cruz,
Santa Cruz, CA, 95064
mcd.ucsc.edu/faculty/ward.html
Correspondence to: Jordan D. Ward (jward2@ucsc.edu)
I am delighted to announce that as of Jul 1st, 2016 the Ward lab has opened at the University of California, Santa Cruz in the Department of Molecular, Cellular, and Developmental Biology. We are interested in how the remarkable complexity and noise of gene regulation is converted into the beautiful and precise cellular behaviors that drive animal development. We use a combination of genetics, molecular biology, microscopy, in vitro biochemistry, and genomics to determine how individual, evolutionarily conserved transcription factors regulate distinct gene expression programs controlling cell division and differentiation and organ development in living animals. We are especially interested in transcription factor regulation of two cellular processes: i) development of the nematode vulva, which is a paradigm of organogenesis; and ii) the nematode molt, which is the shedding of the old skin (cuticle) and generation of a new one. We also extend our findings into the human parasitic nematode Brugia malayi, which causes the disfiguring disease lymphatic filariasis, in an effort to understand how the gene regulatory networks evolve in the context of a parasitic life cycle. For more information, visit our lab website: http://www.jordandward.com

 

Positions are available at all levels for enthusiastic, collaborative scientists interested in both basic research and translating these findings to combating neglected tropical diseases. Write to Jordan D. Ward if interested: jward2@ucsc.edu

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The daf-2(e1370) allele is not temperature sensitive, however, the dauer phenotype is temperature sensitive

Collin Yvès Ewald1,2 and T. Keith Blackwell2
1 Eidgenössische Technische Hochschule (ETH) Zürich, Health Sciences and Technology, Schwerzenbach-Zürich, Switzerland
2 Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, United States
Correspondence to: T. Keith Blackwell (keith.blackwell@joslin.harvard.edu)
The daf-2 reference allele e1370 is used widely to assess the function of reduced Insulin/IGF-1 receptor signalling in development, metabolism, stress-defence, and aging. The daf-2(e1370) mutants develop into dauers at higher temperatures (25°C) but not at lower temperatures (15°C; (Gems et al. 1998)). Based on this observation, the assumption is that daf-2(e1370) mutants have lower insulin/IGF-1 signalling (IIS) at 25°C and presumably normal or wild-type IIS at 15°C. However, two pieces of evidence argue against this assumption.

  1. At lower temperature (15°C), the daf-2(e1370) mutants are long-lived to the same extent as at higher temperatures (Figure 1). In addition, at lower temperature (15°C) daf-2(e1370) mutants are also stress resistant (Gems et al. 1998).
  2. The transcription factors DAF-16 and SKN-1 that are phosphorylated directly by IIS, and thereby prohibited from entering the nucleus, are nuclear in daf-2(e1370) mutants at 15°C to the same extent as at higher temperatures (Lin et al. 2001 PMID:11381260; Tullet et al. 2008; Ewald et al. 2015).

These findings suggest that IIS is reduced comparably at 15°C and 25°C and argues against the assumption that e1370 is necessarily a temperature-sensitive allele in the classic sense where temperature affects protein folding and activity. Interestingly, about 10% wild-type worms form dauers when grown at 27°C under otherwise standard culturing conditions (ample food and not overcrowded; (Ailion & Thomas 2000 PMID:11063684)). Furthermore, it has been observed that null mutants in other abnormal-DAuer-Formation (daf) genes than daf-2, only show the dauer phenotype at higher temperatures, suggesting that the dauer phenotype itself is temperature sensitive. This observation was published over 30 years ago (Golden & Riddle 1984) and is discussed in (Wood 1988).

We are motivated to write to the WBG community by the assumption that during adulthood daf-2(e1370) mutants improperly reactive phenotypes that are reminiscent of dauer larvae (reduced mobility, reduced brood-size, short body size; (Gems et al. 1998)). Recently, we have shown that daf-2(e1370) mutants form dauers independent of SKN-1/NRF1,2,3 and that SKN-1 is only required for the daf-2(e1370)-longevity phenotype when these dauer-related phenotypes are not activated during adulthood (Ewald et al. 2015). Furthermore, comparing expression profiles of daf-2(e1370) mutants with dauer larvae revealed that they share only a common overlap under conditions that reactivate these dauer phenotypes (e.g., daf-2(e1370) at higher but not at lower temperatures; (Ruzanov et al. 2007 PMID:17543485; McElwee et al. 2004 PMID:15308663; Ewald et al. 2015)). Finally, treating wild-type worms with dauer pheromone was sufficient to increase lifespan (Kawano et al. 2005 PMID:16377915; Ewald et al. 2015). This suggests that there might be a dauer program activated in daf-2(e1370) mutants at higher temperatures that masks other underlying mechanisms of reduced IIS on lifespan. Further research is required, however, these findings suggest that reduced IIS can extend lifespan via dauer-dependent and dauer-independent mechanisms and this changes the interpretation of experiments performed with daf-2(e1370) mutants under conditions that promote these dauer-associated phenotypes.

Figures



Figure 1: daf-2(e1370) mutants are long-lived at lower temperature

References

Ewald, C.Y. et al., 2015. Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity. Nature, 519(7541), pp.97–101.  PubMed

Gems, D. et al., 1998. Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics, 150(1), pp.129–155.  PubMed

Golden, J.W. & Riddle, D.L., 1984. A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proceedings of the National Academy of Sciences of the United States of America, 81(3), pp.819–823.  PubMed

Tullet, J.M.A. et al., 2008. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell, 132(6), pp.1025–1038.
  PubMed

Wood, W.B. ed., 1988. The Nematode Caenorhabditis elegans. 1st ed., Cold Spring Harbor Monograph Series.  

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