Fire A et al. (1990) Nucleic Acids Res "Vectors for low copy transformation of C. elegans."

Kondo K, Waterston RH, Fire A

[Nucleic Acids Res, 1990]

The amber suppressor gene sup-7 has been used as a selectable marker to introduce cloned DNA at low copy number into C. elegans chromosomes. We describe structure and several characteristics of seven new sup-7 containing transformation vectors.

Cornwell AB et al. (2020) Methods Mol Biol "Analysis of Lifespan in C. elegans: Low- and High-Throughput Approaches."

Samuelson AV, Cornwell AB

[Methods Mol Biol, 2020]

Lifespan is the most straightforward surrogate measure of aging, as it is easily quantifiable. A common approach to measure Caenorhabditis elegans lifespan is to follow a population of animals over time and score viability based on movement. We previously developed an alternative approach, called the Replica Set method, to quantitatively measure lifespan of C. elegans in a high-throughput manner. The replica set method allows a single investigator to screen more treatments or conditions in the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. Unlike traditional approaches, the Replica Set method centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with "traditional" longitudinal methods. The protocols provided here describe both the traditional experimental approach and the Replica Set method, as well as practical considerations for each.

Huaqi Jiang et al. (2001) International C. elegans Meeting "hif-1, a homolog of mammalian hypoxia-inducible factor-1 alpha, is required for adaptation to low oxygen in C elegans"

Jo Anne Powell-Coffman, Rong Guo, Huaqi Jiang

[International C. elegans Meeting, 2001]

Hypoxia- inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development and plays a critical role in tumor progression. We have determined that a similar complex mediates the response to hypoxia in C. elegans . This complex consists of HIF-1 and AHA-1, two proteins that contain basic-helix-loop-helix and PAS domain motifs. hif-1 is the C. elegans homolog of mammalian HIF-1 a. We screened for deletions in hif-1 and isolated hif-1 (ia04) , a 1,231bp deletion of the second, third, and forth exons that results in a shift of frame. hif-1 (ia04) worms exhibit no visible defects under standard laboratory conditions, but they are unable to adapt to low levels of oxygen. While wild type animals survive and reproduce in 1% oxygen, 73% of hif-1 -defective animals die in these conditions. The hif-1 promoter directs expression of GFP in all somatic cells. However, the expression of HIF-1 is post-transcriptionally regulated. T he level of HIF-1:GFP increases in hypoxic conditions, and the fusion protein is rapidly degraded upon re-oxygenation. HIF-1 can be co-immunoprecipitated with AHA-1. aha-1 is the C. elegans ortholog of mammalian ARNT. In addition to its role in hypoxia response, aha-1 has essential functions during embryogenesis and larval development. We propose that the mechanisms of hypoxia signaling are conserved among metazoans and that C. elegans is an excellent model system for studying hypoxia signaling and response. Additionally, we find that nuclear localization of AHA-1 is disrupted in a hif-1 mutant. This suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.

Barrett A et al. (2021) G3 (Bethesda) "A head-to-head comparison of ribodepletion and polyA selection approaches for C. elegans low input RNA-sequencing libraries"

Taylor SR, McWhirter R, Barrett A, Hammarlund M, Miller DM, Weinreb A

[G3 (Bethesda), 2021]

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

Andrew Papp et al. (2001) International C. elegans Meeting "The Low-Cost Worm Lab"

William Rhoades, Dan Houchin, Andrew Papp

[International C. elegans Meeting, 2001]

The equipment for Molecular Biology and Genetics research traditionally has been expensive, due to a variety of factors, including: low production volumes, high precision requirements, and complex sales/distribution systems. We have tried to improve this situation through the use of innovative or alternative technologies, and the adaptation of consumer and industrial mass-produced equipment for use in the biology laboratory. While a variety of approaches have been pursued toward the efficient DNA-mediated transformation of C. elegans (Papp et al., 1997; Wilm et al. 1999), so far, no method has rivaled the effectiveness of direct microinjection (Mello et al., 1995). A major "difficulty" with microinjection is the cost of an inverted microscope with Differential Interference Contrast (DIC) optics and a Microinjection Controller. In response to this, we developed a low-cost electronic foot pedal controlled Microinjection Controller (Papp et al., 1991), and more recently, coupled it to a low-cost inverted microscope that utilizes custom-made Hoffman Modulation Contrast (HMC) optics, along with an inexpensive glide stage and micromanipulator. These high numerical aperture HMC optics yield a DIC-like image of oocytes and syncytial nuclei. While not ideal for demanding cell lineage studies, this system is more than adequate for microinjection. We can retail our complete system for $10,000, about one-third the cost of traditional systems, making it a nice dedicated microinjection station. Proper maintenance of C. elegans cultures, particularly temperature-sensitive mutants, requires a precisely temperature-controlled incubator. Incubators with the ability to both heat and cool are traditionally expensive devices employing both Freon compressors and heating coils that fight against each other to control the temperature. In response to this, we developed a low-cost microprocessor-controlled incubator, based on the Peltier thermoelectric effect, which is now commonly employed in consumer-grade coolers. This unit quickly learns just the right amount of power to apply to the heat pump, in order to maintain the set temperature with about 0.1 degree Celsius precision, and it has sophisticated alarms to inform the user if anything has gone wrong. These incubators are available for less than $800, about one-third the cost of traditional heating and cooling incubators. C. elegans genetics experiments require many media-filled Petri dishes. Traditional Sterile Media Dispensers utilize unreliable peristaltic pumps, or unreliable syringe based pumps, both of which are expensive and often cumbersome. In response to this, we developed a low-cost, all-digital microprocessor-controlled Sterile Media Dispenser based on a miniature autoclavable electromagnetic pump. This unit has a variety of advantages included memory storage for common sizes of Petri dishes, and retails for $650, about one-third the cost of other Sterile Media Dispensers. Samples of this equipment, as well as a discussion of ideas in the pipeline will be at the Poster Session. Mello C, Fire A. 1995. DNA transformation. "Methods in Cell Biology. Caenorhabditis elegans : Modern Biological Analysis of an Organism." H.F. Epstein and D.C. Shakes (eds), Academic Press, Inc., San Diego. 48: 451-482. Papp A, Lee J, Farris K. 1997. WORM ZAPPING II: C. ELEGANS ELECTROPORATION? 1997 International Worm Meeting abstract 466. Papp A, Mancillas J. 1991. Improved Injection Setup! Worm Breeder's Gazette 12(1): 52. Wilm T, Demel P, Koop HU, Schnabel H, Schnabel R. 1999. Ballistic transformation of Caenorhabditis elegans . Gene 229: 31-35.

Wu, Ziyun et al. (2017) International Worm Meeting "An essential role for p38/ATF-7 pathway in survival under low potassium environment in C. elegans."

Blackwell, T. Keith, Wu, Ziyun

[International Worm Meeting, 2017]

Potassium ions are essential for all living cells. In humans, potassium levels have critical effects on action potentials, blood pressure, osmotic pressure, acid-base balance, metabolism and many other health parameters. Although C. elegans has frequently been employed to study the nervous system, genetics, development, metabolism and aging, how potassium levels affect C. elegans survival has not been investigated, the molecular mechanisms in regulation of survival in response to low potassium environment are unknown. Here we determined that the conserved p38 MAPK signaling pathway NSY-1(ASK1)- SEK-1(MKK3)-PMK-1(p38) is essential for C. elegans survival under low potassium conditions. Wild-type worms are not sensitive to low potassium environment and able to survive more than one month. By contrast, under these conditions animals in which this p38 pathway is disabled become paralyzed within two hours, and die within a few days. Through EMS mutagenesis screening of sek-1(km4) animals for resistance to low potassium, we determined that the p38 pathway mediates resistance to low potassium by regulating the transcription factor ATF-7. Additionally, pharmacological studies suggest that A-type K+ channels are critical for survival under low potassium conditions. The essential function of sek-1 in resistance to low potassium can be rescued by its expression specifically in the ASI chemosensory neurons and not the intestine, revealing that this function is distinct from the roles of sek-1 and p38 signaling in dietary restriction and pathogen resistance. Further genetic analyses in C. elegans may be invaluable for unraveling neuronal tissue-nonautonomous signaling mechanisms that maintain potassium homeostasis.

Wahlby C et al. (2014) Methods "High- and low-throughput scoring of fat mass and body fat distribution in C. elegans."

Wahlby C, Kamentsky L, Larkins-Ford J, O'Rourke EJ, Sokolnicki KL, Michaels K, Veneskey M, Bray MA, Carpenter AE, Lee-Conery A

[Methods, 2014]

Fat accumulation is a complex phenotype affected by factors such as neuroendocrine signaling, feeding, activity, and reproductive output. Accordingly, the most informative screens for genes and compounds affecting fat accumulation would be those carried out in whole living animals. Caenorhabditis elegans is a well-established and effective model organism, especially for biological processes that involve organ systems and multicellular interactions, such as metabolism. Every cell in the transparent body of C. elegans is visible under a light microscope. Consequently, an accessible and reliable method to visualize worm lipid-droplet fat depots would make C. elegans the only metazoan in which genes affecting not only fat mass but also body fat distribution could be assessed at a genome-wide scale. Here we present a radical improvement in oil red O worm staining together with high-throughput image-based phenotyping. The three-step sample preparation method is robust, formaldehyde-free, and inexpensive, and requires only 15min of hands-on time to process a 96-well plate. Together with our free and user-friendly automated image analysis package, this method enables C. elegans sample preparation and phenotype scoring at a scale that is compatible with genome-wide screens. Thus we present a feasible approach to small-scale phenotyping and large-scale screening for genetic and/or chemical perturbations that lead to alterations in fat quantity and distribution in whole animals.

Chen YL et al. (2019) Nat Commun "Adiponectin receptor PAQR-2 signaling senses low temperature to promote C. elegans longevity by regulating autophagy."

Zhao PJ, Xu JP, Chen YL, Zhang KQ, Tao J, Zou CG, Tang W

[Nat Commun, 2019]

Temperature is a key factor for determining the lifespan of both poikilotherms and homeotherms. It is believed that animals live longer at lower body temperatures. However, the precise mechanism remains largely unknown. Here, we report that autophagy serves as a boost mechanism for longevity at low temperature in the nematode Caenorhabditis elegans. The adiponectin receptor AdipoR2 homolog PAQR-2 signaling detects temperature drop and augments the biosynthesis of two -6 polyunsaturated fatty acids, -linolenic acid and arachidonic acid. These two polyunsaturated fatty acids in turn initiate autophagy in the epidermis, delaying an age-dependent decline in collagen contents, and extending the lifespan. Our findings reveal that the adiponectin receptor PAQR-2 signaling acts as a regulator linking low temperature with autophagy to extend lifespan, and suggest that such a mechanism may be evolutionally conserved among diverse organisms.

Gennifer E. Merrihew et al. (2006) European Worm Meeting "A Mass Spectrometry Method to Detect and Quantitate Low Abundance Proteins in a C. elegans"

Gennifer E. Merrihew, Michael J. MacCoss

[European Worm Meeting, 2006]

Gennifer E. Merrihew and Michael J. MacCoss. The detection of low abundance proteins in complex mixtures can be a tedious, expensive and time-consuming ordeal. Current approaches rely on the use of antibodies to detect the protein of interest by Western blotting. An alternative approach is the mass spectrometry equivalent of a Western blot. In this approach, a unique tryptic peptide(s) for a protein of interest is monitored by high resolution selected reaction monitoring (SRM) mass spectrometry, and the peptide precursor ion, fragment ion, and retention time facilitate the detection and quantitation of the protein in a mixture. We applied this technique to the C. elegans proteins DAF-16, a FOXO transcription factor and AKT-1, a serine/threonine kinase, both of which are essential components of the C. elegans insulin/IGF-1 signaling pathway. To test the detection of DAF-16 and AKT-1 in C. elegans lysate, 77 ug of total protein was injected per LC-MS/MS analysis. We were able to easy detect both endogenous DAF-16 and AKT-1 with a short 15 min chromatography gradient. Additionally, we can measure the absolute protein abundance using standard addition of synthetic peptide standards of DAF-16 and AKT-1. We will present experimental confirmation of both the detection and quantitation of both of these proteins. Also, we will validate our mass spectrometry method by directly comparing traditional Western blots and mass spec westerns.

Wood WB et al. (1996) Dev Genet "Maternal effect of low temperature on handedness determination in C. elegans embryos."

Florance A, Wood WB, Bergmann DC

[Dev Genet, 1996]

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16 degrees C or 20 degrees C under standard conditions. However, among the progeny of adults reproducing at 10 degrees C, the frequency of animals with sinistral handedness was increased to approximately 0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10 degrees C and mated with males reared at 20 degrees C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component.