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A novel null allele of C. elegans gene ceh-14

Emily A. Bayer & Oliver Hobert
We have generated a novel null allele, ot900, of the C. elegans LIM homeodomain gene ceh-14. All existing deletion alleles of ceh-14 were generated using a Tc1 transposon insertion, one of which, ch3, is a putative null based on the resulting frame-shift and a reduction in detectable mRNA and protein levels (Cassata et al. 2000). However, in our hands we have noticed a propensity of the ch3 allele to revert to wild-type after several generations,...

Loss of eif-2alpha phosphorylation on S49 (mammalian S51) associated with the integrated stress response hastens development in C. elegans​

Jarod Rollins, Noah Lind & Aric N. Rogers
The gene eif-2alpha (Y37E3.10) in C. elegans encodes for the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). The eIF2 trimer is required for delivering charged tRNAMet to the ribosome in a reaction requiring the hydrolysis of GTP to GDP (Sokabe et al., 2012). The phosphorylation of eif2-alpha on its 49th serine residue (Nukazuka et al., 2008) prevents replenishing GDP with GTP and thus reduces translation initiation by preventing delivery of tRNAMet. The phosphorylation...

Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells

Sierra K. Lear, Alakananda Das & Miriam B. Goodman
Four transgenes designed to drive expression of Cre recombinase and a nuclear-localized blue fluorescent protein (::Cre::F2A::BFP::NLS::tbb-2 3'UTR) were expressed under the control of neuron-specific promoters. All transgenes were inserted into the ttTi5605 transposon insertion site in chromosome II of wild-type C. elegans animals (N2) using CRISPR/Cas9-mediated genome editing, equalizing any potential positional effects. We observed very faint BFP signal in live worms from the signal copy transgene insertions. Additionally, the signal was further masked by...

Novel deletion alleles of a C. elegans gene Y48E1C.1, named as tm5468, tm5625 and tm5626

S; Suehiro Hori
We report tm5468, tm5625 and tm5626 as novel deletion alleles of the gene Y48E1C.1 that is the only ortholog of human calmodulin-lysine N-methyltransferase (CAMKMT)1. CAMKMT encodes an evolutionarily conserved enzyme class I protein methyltransferase that acts in the formation of trimethyllysine in calmodulin for calcium-dependent signaling2. CAMKMT mutation is associated with Hypotonia-cystinuria syndrome in human2,3. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV4. In the screening, all the alleles...

daf-2 modulates regeneration of mechanosensory neurons I

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
Caenorhabditis elegans (C. elegans) possess the ability to spontaneously regenerate injured axons via a highly efficient mechanism known as axonal fusion (Ghosh-Roy, et al., 2010; Neumann et al., 2011; Neumann et al., 2015; Abay et al., 2017). Following laser axotomy, regrowth from the proximal axon segment (still attached to the cell body) reconnects and fuses with its separated distal segment (Fig. 1A). We recently demonstrated that the level of axonal fusion increases with age (Abay...

Expression pattern of endogenous PAR-4A & C after CRISPR/Cas9-mediated genome editing

Vincent Roy, Olivier Gagné, Karim Hamiche, Jean-Claude Labbé & Patrick Narbonne
PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al....

Delta-9 Fatty Acid Desaturase Mutants Display Increased Body Size

James F.; Savage-Dunn Clark
C. elegans contain three Δ9 fatty acid desaturases (FADs), fat-5, fat-6, and fat-7, responsible for the initial desaturation step in the synthesis of complex polyunsaturated fatty acids (PUFAs). Production of PUFAs is vital for survival, as simultaneous loss-of-function in all three genes results in synthetic lethality (Brock et al. 2006). Animals containing mutations in any one of the three Δ9 FADs, fat-5, fat-6, and fat-7, display an increase in body length. fat-5 mutants display a...

Endocannabinoid 2-arachidonoyl glycerol increases the transcription of daf-7 in ASI neurons

Celina Galles, Gastón M Prez & Diego De Mendoza
daf-7 expression in ASI neurons is increased when nematodes are grown in the presence of endocannabinoid 2-arachidonoyl glycerol (2-AG). Left upper panel: representative image of reporter strain FK181 ksIs2 [Pdaf-7::GFP+rol-6(su1006)] captured under GFP epifluorescence microscopy. Right upper panel: same individual showing bright field microscopy together with GFP signal. Gray/black triangles mark ASI neurons expressing daf-7. Scale bars correspond to 0.03 mm. Lower panel: point plot of the adjusted GFP intensity/ASI neuron values gathered from worms...

New allele of C. elegans gene spch-3 (T27A3.4), called xc2

Nicholas R. Munoz, Dana T. Byrd & Diana Chu
We created a new mutant allele, named xc2, of the gene spch-3 (T27A3.4). spch-3 encodes one of three C. elegans small, highly basic proteins that resemble invertebrate protamines and associate with sperm meiotic chromosomes (Chu et al., 2006). The alleles were isolated from gene mutations generated by Non-Homologous End Joining (NHEJ) mediated repair of Cas9-generated breaks (Dickinson et al., 2013; Ran et al., 2013). The alleles were discovered using PCR with the following primers 5’-...

Efficient FLP-mediated germ-line recombination in C. elegans

Javier Macías-León & Peter Askjaer
​Recombinases are very useful enzymes for spatiotemporal control of gene expression (Hubbard 2014). Through binding to specific DNA sequences, they can be used to excise or exchange DNA fragments. We have previously generated a series of Caenorhabditis elegans strains that stably express an efficient FLP recombinase in specific tissues and reported how they enable precise regulation of gene expression, cell ablation or conditional gene knockout (Muñoz-Jiménez et al 2017). However, these strains are restricted to...

Localization of tbg-1 mRNAs and GFP::TBG-1 protein in Early C. elegans Embryos

Lauren DeMeyer & Mi Hye Song
tbg-1 encodes gamma-tubulin, a ubiquitous and highly conserved component of centrosomes in eukaryotic cells (Strome et al, 2001). Using smFISH we determined the localization of tbg-1 transcripts (red). tbg-1 transcripts are detected within distinct foci throughout the cytoplasm during both the first (A) and second (B) mitosis. tbg-1 transcripts are not enriched at centrosomes or either blastomere. In contrast, GFP tagged TBG-1 proteins (green signal; arrowheads) localize at centrosomes, as previously shown (Strome et al,...

Reduced pharyngeal pumping rates observed in tph-1 mutants using microfluidic electropharyngeogram (EPG) recordings

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In Caenorhabditis elegans, serotonin (5-HT) activates and controls pharyngeal pumping in response to food (Horvitz et al., 1982; Sze et al., 2000; Song and Avery 2012). Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping (Sze et al., 2000; Avery and Horvitz 1990; Song and Avery 2012). We used a microfluidic electropharyngeogram...

The protein kinase G orthologs, EGL-4 and PKG-2, mediate serotonin-induced paralysis of C. elegans

Andrew C. Olson & Michael R. Koelle
The C. elegans protein kinase G ortholog, EGL-4, has been demonstrated to be involved in C. elegans egg laying and the initiation of dwelling, a behavioral state characterized in part by low rates of locomotion (Trent et al. 1983; Hao et al. 2011). Serotonin regulates C. elegans egg laying through all five identified serotonin receptors, and serotonin promotes the dwelling state through the mod-1 serotonin receptor (Trent et al. 1983; Hapiak et al. 2009; Flavell...

Basal pharyngeal pumping elevated in C. elegans mod-5 mutants

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In C. elegans, the reuptake of serotonin (5-HT) is facilitated by mod-5, which encodes a 5-HT transporter that is orthologous to a human 5-HT transporter (SLC6A4). mod-5 has been shown to effect both feeding and locomotion in C. elegans (Ranganathan et al., 2001; Jafarau et al. 2011). We obtained and analyzed EPG data using a microfluidic device (NemaMetrix) for mod-5 null mutant strains, mod-5(n3314) (A, Exp 1 n=15; Exp 2 n=32) and mod-5(n822), (B, Exp...

wrt-2 expression oscillates during larval development

Ana G. Vera-Cruz, Siavash Amon, Sabih Rashid & Lesley T. MacNeil
wrt-2 encodes a hedgehog-like protein that is weakly required for molting (Kouns et al., 2011). As monitored using a transcriptional GFP reporter, wrt-2 is expressed in seam and hypodermal cells throughout development (Apock et al., 1999). However, wrt-2 transcript levels oscillate with a period consistent with the timing of the molting cycle (Hendriks et al., 2014). Because standard GFP reporters do not capture rapid changes in gene expression, we generated a wrt-2 transcriptional reporter driving...

The Effect of an Environmentally Relevant Phthalate Mixture on Primordial Germ Cells of Xenopus laevis Embryos

Nathalie L Momplaisir, Aurora Turgeon, Jodi Flaws & Jing Yang
Phthalates, being considered endocrine disrupting chemicals, are estrogen-like and anti-androgenic compounds that interfere with hormones which regulate numerous biological processes, including the maintenance of homeostasis and the regulation of development (Kavlock et al., 1996). Toxicological studies have consistently shown that phthalate metabolites are reproductive and developmental toxicants (Jeng, 2014). Some of the significant complications include fertility issues, decreased anogenital distance, low sperm concentration and motility. While the adverse effects of phthalates on the reproductive system...

Novel deletion alleles of a C. elegans gene C38D4.9, named as tm4476 and tm4561

Sawako Yoshina, Sayaka Hori, Yuji Suehiro & Shohei Mitani
We report tm4476 and tm4561 as novel alleles of C38D4.9 gene that is an ortholog of human METTL5 (methyltransferase like 5)1. Human METTL5 function has not been reported. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV2. In the screening, both the alleles were detected by nested PCR using the following primer sets, 5’-CCGCCTATATCATGGCGCTT-3’ and 5’-GCTGGTAGTTGCCACCGCAT-3’ for first round PCR and 5’-ACTATCGAAGCCGCGCTTCA-3’ and 5’-AACCCCAACTCCGTCCCATT-3’ for second round PCR. By Sanger...

Genetic mapping of EgfrL.3.1 in Drosophila melanogaster

Joyce Stamm, Gnanada S Joshi, MA Anderson, Katie Bussing, Colton Houchin, Amber C Elinsky, Jacob T Flyte, Nadine Husseini, Dominika Jarosz, Chelsea L Johnson, Abby F Johnson, Christina E Jones, Taj P Kooner, Daniel Myhre, Thomas N Rafaill, Sarah Sayed, Kirby W Swan, Jonathan Toma & Jacob D Kagey

daf-2 modulates regeneration of mechanosensory neurons II

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
The daf-2 gene encodes an insulin-like growth factor/IGF-1 receptor that regulates C. elegans embryonic and larval development. It has previously been shown that DAF-2 inhibits neurite regeneration of the GABAergic motor neurons and PVD sensory neurons in an age-dependent fashion (Bryne et al., 2014; Kravtsov et al., 2017). Following injury, the posterior lateral microtubule (PLM) neurons are capable of regenerating through axonal fusion, a highly efficient regrowth mechanism in which separated fragments fuse back together...

A quantitative trait locus for nictation behavior on chromosome V

Jun Kim, Daehan Lee & Junho Lee
Lee et al., 2017 showed that CB4856 has a lower nictation ratio (the number of nictating dauers over the number of moving dauers on a micro-dirt chip) than N2, and that a genetic locus (nict-1) on chromosome IV mediates this phenotype difference. Although this paper had no evidence of any other genetic loci for nictation behavior, we have studied other quantitative trait loci (QTL) using different linkage-based mapping strategies. Here, we report another genetic locus...

Chemotaxis based enrichment for transgenic animals containing the rol-6 marker

John L. Carter, Rafael Morales & Steven M. Johnson
Utilizing the chemotaxis of C. elegans, we can separate wild-type C. elegans from “rollers.” If a mixed population of transgenic worms harboring the rol-6 marker, and their non-transgenic siblings are plated a distance from food, “rollers” will be unable to efficiently migrate towards the food due to impaired locomotion, while non-transgenic C. elegans will have normal locomotion and be able to quickly migrate to the food source. This will efficiently separate the two types of...

An isoform-specific allele of the sax-7 locus

Dylan Rahe, Ines Carrera, Filip Cosmanescu & Oliver Hobert

SapTrap vectors for introducing point mutations with unc-119+ selection

Matthew L Schwartz & Erik M Jorgensen
SapTrap assembly generates CRISPR targeting vectors using a Golden Gate cloning strategy (Schwartz and Jorgensen, 2016). The original toolkit was designed for inserting tags, such as fluorescent proteins or affinity tags, into protein coding genes in C. elegans. In the original design, the floxed unc-119+ selectable marker was incorporated into the tag. For site-directed mutagenesis, the selectable marker is useful but a tag is unnecessary. We have developed a new set of vectors dedicated for...

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