Rodrigues, Pedro Reis et al. (2009) International Worm Meeting "A variety of age-dependant aggregating proteins determine lifespan."

Peters, Theodore, Gibson, Brad, Lithgow, Gordon, Hughes, Robert, Alavez, Silvestre, Rodrigues, Pedro Reis, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Protein aggregation has for long been hypothesised as a determinant of lifespan. Briefly, normal cellular activity may give rise to damaged proteins causing them to become insoluble, missfold and aggregate. To test this hypothesis we adapted a protocol in order to extract insoluble proteins from synchronously aging populations of C. elegans. Proteins were separated based on their aqueous and detergent solubility and the insoluble fraction was resolubilized in 70%; formic acid. Insoluble proteins were chemically labelled, identified and quantified by liquid chromatography coupled with mass spectrometry (LC- ESI-MS/MS). We identified a range of proteins with roles in various cellular processes and possibly from a range of cellular compartments. 27%; of the proteins identified as forming aggregates have previously been shown to be important in keeping low levels of polyglutamine aggregation1. This suggests that reduced soluble levels of these proteins caused by age-related aggregation may cause increased risk of polyglutamine aggregation. We then considered whether proteins that appear to form aggregates during normal aging influenced lifespan. To test this notion we, reduced their expression in adult animals (from 4 days old) by RNA interference (RNAi). 34%; of the RNAi treatments were found to significantly extend mean lifespan in C. elegans suggesting that a variety of age-dependant aggregating proteins determine lifespan. Among the insoluble proteins, DAF-21, an ortholog of the mammalian HSP-90, showed age-dependant aggregation and is being used as a marker to study the role of several molecular pathways in protein aggregation. Taken together our results suggest that protein aggregation may play a common and key role in aging and age-related disease. 1 - Nollen, E., Garcia, S., Haaften, G., Kim, S., Chavez, A., Morimoto, R., Plasterk, R., Genome-wide RNA interference screen identified previously undescribed regulators of polyglutamine aggregation. Proc Natl. Acad. Sci., 101, 6403-6408, 2004.

Lucanic, Mark et al. (2009) International Worm Meeting "Endocannabinoid signaling has a role in dauer formation."

Held, Jason, Lucanic, Mark, Gibson, Brad W, Lithgow, Gordon J, Gill, Matthew, White, Mark, Klang, Ida

[International Worm Meeting, 2009]

The endocannabinoid system in mammals plays a role in many physiological functions and is an important emerging therapeutic target. Cannabinoid receptors have been identified in many different species, including some invertebrates, but they have not been detected in C. elegans. We have now identified components of an endocannabinoid system in C. elegans and find that it interacts with the insulin signaling pathway to modify dauer formation. In a chemical screen for suppressors of dauer formation we identified a synthetic antagonist of the mammalian cannabinoid receptor as a potent suppressor of the Daf-c phenotype of daf-2(e1368). The activity of this compound, and other synthetic cannabinoids, prompted us to take a biochemical approach to defining a worm endocannabinoid system, with a focus on identifying endogenous cannabinoid ligands (endocannabinoids). Analysis of worm lipid extracts by gas chromatography-mass spectrometry (GC-MS) identified a number of fatty acid ethanolamides that are structurally similar to mammalian endocannabinoid ligands. We find that endocannabinoid levels are reduced in daf-2(e1368) mutants under dauer inducing conditions and that endocannabinoid treatment is sufficient to promote reproductive growth in these animals, similar to the effects of synthetic cannabinoids. We also find that levels of endocannabinoids are elevated following treatment with a chemical inhibitor of fatty acid amide hydrolase (FAAH), an enzyme involved in endocannabinoid degradation and inactivation, and that RNAi of the putative worm FAAH gene has a similar effect. Overexpression of this putative C. elegans FAAH gene in wild type animals results in a profound developmental delay, further supporting a role for endocannabinoids in reproductive growth. Finally, daf-2 mutants carrying extra copies of this FAAH gene under heterologous transcriptional control are defective in dauer exit, suggesting a role for endocannabinoids in dauer recovery. In summary these data suggest that endocannabinoids function in C. elegans to promote reproductive growth either under normal conditions or following recovery from dauer arrest. The lack of obvious cannabinoid receptors in the worm suggest the existence of a novel C. elegans receptor, whose identity may shed light on the novel receptors predicted to exist in mammals. Finally the identification of an endocannabinoid system in nematodes now brings the strengths of C. elegans as a major model system to the study of endocannabinoid physiology.

Rogers, Aric N. et al. (2009) International Worm Meeting "eIF-4G post-transcriptionally remodels stress response and cellular homeostasis gene expression in C. elegans."

Chen, Di, Hubbard, Alan, McColl, Gawain, Kapahi, Pankaj, Lithgow, Gordon, Rogers, Aric N., Melov, Simon, Gibson, Brad, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Attenuation of mRNA translation extends lifespan in metazoans by as yet unknown mechanisms. Eukaryotic translation initiation factor (eIF)-4G is an mRNA cap-binding complex member that positively regulates translation by acting as a scaffold between transcript 5'' and 3'' ends via eIF-4E and poly A binding protein, respectively. Here we show that reducing translation via suppression of eIF-4G (ifg-1 in C. elegans) also differentially regulates expression of genes involved in responding to stress. Furthermore, we observe a positive correlation between mRNA size and translation as measured by ribosome loading when ifg-1 is inhibited. This correlation extends to overall protein levels assayed using quantitative mass spectrometry. Genome-wide analysis shows that longer mRNAs are enriched with genes important for stress responses and that positively regulate lifespan and cellular homeostasis. Inhibition of some of these stress response genes substantially diminishes the increased lifespan under eIF-4G inhibition and include the FOXO transcription factor daf-16 and the energy sensor AMP kinase catalytic subunit aak-2. Expression of SCA-1, a Calcium ATPase involved in ER homeostasis, is essential for lifespan extension in an ifg-1 mutant. Our results indicate that ifg-1 controls a switch in mRNA translation between growth and development and somatic maintenance, which provides a novel mechanism for the tradeoffs between growth and longevity.

Dickinson DJ et al. (2018) MicroPubl Biol "SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette"

Goldstein B, Dickinson DJ, Chen AH, Slabodnick MM

[MicroPubl Biol, 2018]

The advent of CRISPR/Cas9 technology in C. elegans has enabled an unprecedented level of control over this organisms genome, which is facilitating research in a wide variety of fields. Several different experimental approaches exist for CRISPR in this organism (Dickinson and Goldstein 2016). One particularly powerful approach uses a drug selection with a Self-Excising Cassette (SEC) to isolate the desired genetically-modified animals following Cas9-triggered homologous recombination (Dickinson et al. 2015). The most labor-intensive step in this procedure is the construction of a plasmid-based repair template for homologous recombination. SapTrap (Schwartz and Jorgensen 2016) is a high-throughput cloning procedure that allows modular assembly of repair templates for CRISPR/Cas9-triggered homologous recombination. The original publication that described SapTrap cloning (Schwartz and Jorgensen 2016) used unc-119(+) as a positive selection marker for isolating recombinant strains. Compared to unc-119 selection, SEC selection is faster; can be used directly in a wild-type background; and eliminates an extra injection step followed by outcrossing to remove the selection markers after strain isolation (Dickinson et al. 2015). Because of these advantages of SEC selection over unc-119, we wished to incorporate the SEC into the SapTrap workflow. Here, we describe a toolkit of plasmids that allow building SEC-containing repair templates via SapTrap cloning.In brief, SapTrap links pieces of a repair template construct together by means of unique 3-bp overlaps generated by the type II restriction enzyme SapI. Constructs generated using SapTrap have the general form shown in Figure 1A. In a typical assembly reaction, the sgRNA target is provided as a pair of oligos, the homology arms are provided as PCR products, and a Fluorescent Protein (FP), SEC and linker fragments are provided by pre-existing donor vectors. These fragments are mixed together, along with the requisite enzymes, and are assembled in a single reaction. The linkers can be omitted from the final construct if desired, by designing the homology arms with 3-bp overlaps that anneal directly to the FP or SEC instead of to the linker.Using SapTrap to generate repair templates for SEC selection involves two modifications to the original approach described by Schwartz and Jorgensen. First, an AAG junction is added between the FP and SEC fragments (Figure 1A). Together, these two fragments replace the FP/unc-119(+) donors used by the Jorgensen lab. Second, it is important to ensure that a coding exon follows SEC in order to avoid nonsense-mediated decay. For this reason, we always include the NT-tag linker in our constructs, even for C-terminal tags. We generated and tested fluorescent protein donors covering the current best available fluorescent proteins for C. elegans imaging (Heppert et al. 2016 and our unpublished observations) along with SEC donors containing three different Lox sites that do not recombine with each other, for multiplex editing. These constructs have been deposited at Addgene and are listed in Table 1.Using various combinations of fluorescent protein, SEC and linker donors, we were routinely able to obtain the desired repair templates, although the cloning efficiency was lower compared to the ccdB/Gibson-based cloning approach described by Dickinson et al. (2015) (Figure 1B). Note that, for both the Gibson and SapTrap data reported in Figure 1B, we used PCR products 500-700 bp in length as the homology arms, enabling a direct comparison of the two cloning methods. We have not tested short homology arms formed from annealed oligonucleotides, which were used by Schwartz and Jorgensen (2016), with either cloning approach. Based on our extensive experience with both SapTrap and the ccdB/Gibson-based cloning procedure, we believe that each procedure has advantages. The advantages of SapTrap are:SapTrap is more modular: different fluorophores, epitope tags, and selection cassettes can be freely combined without the need to construct a new FPSEC vector each time.SapTrap does not require the use of ccdB-containing vectors, which are toxic to E. coli and therefore can be difficult to grow and maintain.

Ashley GE et al. (2021) Genetics "An expanded auxin-inducible degron toolkit for Caenorhabditis elegans."

Reiner DJ, Levenson MT, Martinez MAQ, Ragle JM, Dickinson DJ, Davidson BR, Johnson LC, Zhang W, Martinez-Mendez R, Doonan R, Matus DQ, Duong T, Palmisano NJ, Medwig-Kinney TN, Sirota SS, Hibshman JD, Saeger HN, Goldstein B, Ashley GE, Ward JD

[Genetics, 2021]

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Schein JE et al. (1996) West Coast Worm Meeting "CONSTRUCTION AND UTILIZATION OF THE CHROMOSOME IV COSMID TRANSGENIC LIBRARY"

Schein JE, Rose AM, Baillie DL, Franz NW, Janke DL

[West Coast Worm Meeting, 1996]

Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the lin-3 - let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval, lin-3 and let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map; let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido) and let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.

Riddle DL et al. (1984) Worm Breeder's Gazette "Caenorhabditis elegans from Adelaide soil."

Riddle DL, Kirtland SK, Bird AF

[Worm Breeder's Gazette, 1984]

Soil samples (200-300 gms) from the Adelaide area were collected from leafy compost, cultivated garden plots, orchards and open areas at 17 locations. The soil samples were placed over funnel collectors in a misting chamber overnight and the filtrate was collected in 90 ml test tubes. Most of the water was removed by suction, and the remainder was allowed to settle in a conical centrifuge tube. This sediment of worms and debris about 0.1-0.2 ml in volume was transferred to the surface of a 8.5 cm diameter NGM agar petri plate spread with E. coli. After three days incubation at room temperature (24 C), worms which grew and reproduced on the E. coli were selected. Worms morphologically similar to C. elegans were transferred to fresh plates at hourly intervals, 2 to 3 times in succession. These repeated transfers removed contaminating soil microbes. The worms were cloned, and those animals capable of self-fertilization were then mated with C. elegans N2 males. Generally, no more than four clones were made from any soil sample, and two hermaphrodite progeny from each clone were mated with four males on a petri plate. When F1 males (usually 30-60) issued from such a cross, the maternal clone was tentatively identified as C. elegans. Final confirmation was obtained by backcrossing the F1 hybrid males with hermaphrodites from the cloned soil isolate. The appearance of F1 males in this latter cross demonstrated that the hybrid males were fertile. The 17 soil samples produced four new C. elegans isolates: one from Alan Bird's garden (AB1), one from the Australian Wine Research Institute grounds (AB2), and two from Waite Agricultural Research Institute nursery plots. The samples containing C. elegans were moist, well-aerated, and near the surface where decaying vegetable matter provided ground cover. We conclude from these preliminary results that C. elegans may be widespread in the Adelaide area, and perhaps in other areas of Australia as well. Stocks of AB1, AB2, AB3 and AB4 were repeatedly sub-cloned to ensure that a genetically true- breeding stock was obtained. To determine the male bursa morphology exhibited by an Australian C. elegans strain, spontaneous males were picked from a large population (approx. 50,000 worms) of the AB1 clone, and subsequently propagated by mating with AB1 hermaphrodites. The pattern of bursal rays was identical to that previously described for C. elegans isolates from England and France. Southern hybridizations with AB strain genomic DNA using a Tc1 probe (kindly provided by Brad Rosensweig) showed that each of the AB strains is of the low copy number (N2) type, although the AB1 pattern differs slightly from the other three, and they all differ slightly from N2.

Aram, Reta et al. MicroPubl Biol

Li, Chengyin, MacGillivray, Kailynn, Aram, Reta, Saltzman, Arneet L

[MicroPubl Biol, 2019]

Insertion of single-copy transgenes is an important tool in C. elegansgenome engineering. Following the discovery that the Drosophilamariner element Mos1could mobilize in the C. elegansgermline (Bessereauet al.2001), tools for efficient Mos1-mediated transgene insertion have been developed and widely adopted (Robert and Bessereau 2010). In parallel, the establishment of effective antibiotic selection in C. elegansoffered increased versatility for genetic crosses and the advantage of injecting into phenotypically wild-type animals (Giordano-Santiniet al.2010, Sempleet al.2010). Reliable, site-specific, single-copy transgene insertion remains a key technique for applications such as synthetic reporters and rapid testing of genetic rescue constructs, especially when germline expression is required. The highly developed Mos1-mediated single-copy insertion (mosSCI) system is a toolkit of choice for this approach (Frokjaer-Jensenet al.2008, Frokjaer-Jensenet al.2012). The ttTi5605(mos1)II insertion site in particular has been used for stable germline expression, and plasmids targeting ttTi5605are also compatible with the universal mosSCI insertion sites (Frokjaer-Jensenet al.2014). To add to the suite of tools for targeting ttTi5605, we constructed two modified mosSCI plasmids (Figure 1). First, we made a plasmid for ttTi5605-mosSCI using G418 selection. Antibiotic selection has been incorporated into the miniMos system for random transgene insertion (Frokjaer-Jensenet al.2014), however existing ttTi5605-mosSCI plasmids use unc-119(+)selection. Our plasmid allows use of the ttTi5605site without the need for injection into unc-119(ed3)mutants, which are more challenging to grow than wild-type animals and do not have optimal germline health on standard E. coliOP50 as a food source. Second, we modified the standard unc-119selection-based ttTi5605-mosSCI plasmid (pCFJ151) by adding loxP recombination sites flanking the unc-119(+) selection cassette (Figure 1B). This change allows excision of the unc-119(+)selection cassette using an existing Cre recombinase protocol (Dickinson et al. 2013). Since unc-119(+) is a frequently used selection marker, this new feature will add flexibility for construction of strains with multiple transgenes. For increased versatility, both of the plasmids described here are minimal and do not encode additional tags or fluorescent proteins. Multiple-fragment transgenes can be seamlessly fused and cloned into these plasmids using the Gibson isothermal assembly technique by designing PCR amplicons, synthetic dsDNAs, restriction fragments or oligos with overlapping homologous ends (Gibsonet al.2009). We performed three independent test injections using different transgenes and found both of our plasmids to be effective for mosSCI (Figure 1). We also successfully excised the unc-119(+)cassette (Figure 1B) using Cre recombinase for two independent insertions (Methods). However, a caveat of the current NeoR plasmid (Figure 1A) is that no additional phenotypic marker was included between the loxP sites. Therefore, unlike unc-119(+)excision, which can be easily screened for using a visible phenotype, excision of NeoR would require screening by PCR or by loss of drug resistance. Overall, we hope that these excisableunc-119(+)and NeoR mosSCI plasmids will be useful companions to the extensive mosSCI plasmid and strain toolkit (Frokjaer-Jensenet al.2008, Zeiser et al.2011, Frokjaer-Jensen et al.2012, Frokjaer-Jensenet al.2014).

Mendel JE et al. (1990) Worm Breeder's Gazette "WEkan, a New Lambda-Origin Cosmid Vector"

Mendel JE, Sternberg PW

[Worm Breeder's Gazette, 1990]

One is (alas) frequently interested in cloning genes that lie in regions not yet included in the contig map, or in regions spanned by YAC DNA but not by cosmid DNA. Since cloning strategies often involve rescue of mutant phenotypes by injection of cosmid DNAs, both the above situations provide the necessity of chromosomal walking. There are several cosmid vectors designed for chromosomal walking including the lambda-origin vectors such as Lorist6 [Gibson et al., Gene 53:283-286 (1987)] and the ColE1 replicon-based cosmid pWE [Wahl, et al., PNAS 84:2160-2164 (1987)]. These vectors have bacteriophage promoters flanking the cloning site. Chromosomal walking using these vectors consists of making labeled RNA probes from each end of the starting cosmid, determining which probe goes in the desired direction, and using that probe to take the next step. This procedure is facile, but the production of RNA probes can be unreliable. The presence of NotI sites flanking the cloning site in a cosmid vector provides an inexpensive, low-tech way to identify the terminal restriction fragments of a cosmid insert using restriction digests. The terminal fragments, once identified, can be gel isolated and labeled by standard methods. The cosmid vector pWE has NotI sites immediately upstream from the bacteriophage promoters. However, pWE will cross-hybridize with the YAC vector and is therefore not ideal for all applications. Accordingly, we decided to make a lambda-origin vector with the features of pWE. The construction of this vector was straightforward. In brief, Lorist6 has four EcoRI sites, (see below) two of them in essential genes. To protect these sites, Lorist6 was digested with NcoI, end-filled, then redigested with EcoRV. A 2.7 kb restriction fragment containing the cloning site was subcloned into SmaI-EcoRV digested Bluescript regenerating the NcoI site. A 700 bp EcoRI fragment containing the cloning site was removed and replaced with the cloning region of pWE 15, contained in a 100 bp EcoRI fragment. The NcoI-EcoRV fragment, now 2.1 kb, was removed and religated to the 2.5 kb NcoI-EcoRV fragment of Lorist6. The resulting vector was named WEkan, WE from pWE meaning walking easily, and kan because the vector confers resistance to kanamycin. The construction of WEkan resulted in the removal of transcription terminators present in Lorist6 to protect vector genes from transcriptional interference from insert DNA. We do not know what effect this has on representation in libraries prepared in WEkan. It does not seem to affect viability in a general way. [See Figure 1] We prepared a genomic library in WEkan using as insert N2 DNA partially digested with XhoII and size-selected by reverse field gel electrophoresis. Cosmid arms were prepared by digesting with BstEII/BamHI and XbaI/BamHI. DNA prepared from 18 random clones showed that the library contains 10-20% non-recombinant clones and that insert size ranges from 35 to 50 kb. 11,500 members of this library were gridded into micro-titer dishes. The library has been probed with several probes and has been shown to contain three to four genomes. The clones appear to be stable and to grow well.