van der Woude M et al. (2021) STAR Protoc "C.elegans survival assays to discern global and transcription-coupled nucleotide excision repair."

Lans H, van der Woude M

[STAR Protoc, 2021]

Global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) protect cells against a variety of helix-distorting DNA lesions. In <i>C.elegans</i>, GG-NER primarily acts in proliferative germ cells and embryos, while TC-NER acts in post-mitotic somatic cells to maintain transcription. We leverage this difference to distinguish whether proteins function in GG-NER and/or TC-NER by straightforward UV survival assays. Here, we detail a protocol for these assays, using GG-NER factor <i>xpc-1</i> and TC-NER factor <i>csb-1</i> as examples. For complete details on the use and execution of this protocol, please refer to Sabatella etal. (2021).

Roberson ED (2015) PeerJ Comput Sci "Identification of high-efficiency 3'GG gRNA motifs in indexed FASTA files with ngg2."

Roberson ED

[PeerJ Comput Sci, 2015]

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3'GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3'GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3'GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3'GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3'GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.

Laughton DL et al. (1996) Worm Breeder's Gazette "Two Novel Inhibitory Amino Acid Receptors Formed By Alternative Splicing Possess Identical Extracellular Domains"

Lunt GG, Laughton DL, Wolstenholme AJ

[Worm Breeder's Gazette, 1996]

Inhibitory amino-acid receptors, including those for GABA and glutamate, play an important role in the functioning of the nematode nervous system, involved in such activities as locomotion, defaecation and pharyngeal pumping (McIntire et. al, 1993; Avery et. al, 1995; Laughton et. al, 1995). PCR amplifications on C. elegans cDNA using oligonucleotide primers matching conserved regions found in invertebrate GABA-A receptors resulted in the isolation of a number of different partial cDNAs with clear homology to GABA-A receptor subunits. 5' and 3' RACE reactions were used to amplify a full-length cDNA from one of these sequences, named Cegbr 2 (accession no.U40573). This cDNA encodes a protein of 416 amino acids which has all the characteristics of an inhibitory amino acid receptor subunit, including a disulphide-bonded loop in the extracellular N-terminal domain and four hydrophobic, predicted transmembrane regions towards the C-terminus. The spliced leader sequence, SL1, is found at the N-terminus immediately upstream of the start methionine. Interestingly, the 3' untranslated region of this cDNA contains sequence very similar to that encoding the 4 transmembrane regions. During subsequent PCR reactions we isolated an additional cDNA, named Cegbr 3 (ac! cession no. U41113) which encodes a receptor with an identical extracellular domain to Cegbr 2 linked directly to the transmembrane sequence found in the 3'UTR of Cegbr 2 (fig 1). To verify that both transcripts exist in mRNA we performed Northerns using polyA+ RNA isolated from a developmentally mixed population of C. elegans. Radiolabelled cDNA probes specific to the unique transmembrane III-IV intracellular loop sequences of Cegbr 2 or Cegbr 3 were hybridised to duplicate filters and washed under high stringency. As expected, the Cegbr2-specific probe bound to a single transcript of ~2.2Kb (Cegbr 2) while the Cegbr 3 probe bound to two transcripts of ~2.2Kb (Cegbr 2) and ~1.2Kb (Cegbr 3). From these results we predicted that Cegbr 2 and Cegbr 3 are alternatively spliced products of a single gene. To investigate this further we performed genomic PCR across the extracellular/transmembrane boundaries of both subunits. As predicted, both boundaries corresponded with intron splice sites (fig 2). As far as we are aware, this pattern of splicing is unique to nematode fast ion channel receptors, and suggests that these animals will possess receptors with identical ligand binding sites but slightly different channel properties. A similar genomic organisation has been found for a related gene, unc-49, during the C. elegans genome sequencing project. Here, a common N-terminus may be spliced onto three alternative transmembrane domains. This work is supported by the Wellcome Trust.

Laughton DL et al. (1995) Worm Breeder's Gazette "The beta subunit of the avermectin sensitive glutamate receptor is expressed on pm4 pharyngeal muscle cells."

Lunt GG, Laughton DL, Wolstenholme AJ

[Worm Breeder's Gazette, 1995]

The recently cloned avermectin receptor consists of a glutamate sensitive beta subunit and an avermectin sensitive alpha subunit which when co-expressed in Xenopus oocytes lead to the formation of avermectin potentiated glutamate gated chloride ion channels(1). Leon Avery et al have suggested that this receptor mediates the inhibition of pharyngeal muscle contraction via the glutamatergic motor neuron M3(2). Screening of the YAC polytene filter and subsequent cosmids with the 5' end of the beta subunit localised the gene and controlling 5' sequences to cosmid C35E8, which maps to chromosome 1. This cosmid was used as the template for PCR amplification of a DNA fragment consisting of 1426 bp of 5' sequence before the start methionine, plus sequence encoding the first 24 amino acids of the subunit. The fragment was subcloned into the LacZ expression vector, pPD22.11(3) to form a translational fusion. After worm injections we established 6 stably transformed lines which all showed the same beta-galactosidase activity. X-gal staining was present in the 3 pairs of pm4 pharyngeal muscle cell nuclei. No other nuclei consistently stained for X-gal. We have not looked closely at the developmental expression of this subunit but we did see pharyngeal staining in some larval stages (L2/3 onwards) and stained nuclei were seen in some developing eggs. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neuron, and point to inhibition of pharyngeal pumping as a major mode of action for avermectin. It must be remembered however that these experiments have localised the glutamate-binding beta subunit only. The avermectin binding alpha subunit may be expressed in a number of locations , for example body wall muscle, as well as pharyngeal muscle. Acknowledgements We thank Ian Hope for his help with the construction of transgenic worms and Donna Albertson for helping us in the identification of stained nuclei. References (1) Cully et al. (1994) Nature 371: 707-711. (2) Avery et al. (1995) WBG 13(4): 72-73. (3) Fire et al. (1990) Gene 93: 189-198.

Laughton DL et al. (1997) Gene "Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels."

Laughton DL, Lunt GG, Wolstenholme AJ

[Gene, 1997]

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.

Laughton DL et al. (1997) J Exp Biol "Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx."

Laughton DL, Wolstenholme AJ, Lunt GG

[J Exp Biol, 1997]

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

Laughton DL et al. (1995) J Neurochem "The beta-subunit of Caenorhabditis elegans avermectin receptor responds to glycine and is encoded by chromosome 1."

Lunt GG, Wolstenholme AJ, Laughton DL, Wheeler SV

[J Neurochem, 1995]

A full-length cDNA encoding the beta subunit of the recently described avermectin receptor was amplified from Caenorhabditis elegans mRNA. When this cDNA was injected into Xenopus oocytes a dose-dependent response to glycine was observed, together with a smaller response to 1 mM GABA. The EC(50) of the glycine response was similar to that described previously for glutamate (0.38 mM). Hybridisation of the cDNA to polytene filters identified three yeast artificial chromosome clones that gave a positive signal, Y37B3, Y38E5, and Y24C9, all of which are mapped to chromosome 1. Hybridisation to a series of cosmid clones covering this area further mapped the gene encoding this subunit to the region -2,818 to -2,824.

Li BW et al. (2004) Filaria J "Antibody responses to Brugia malayi antigens induced by DNA vaccination."

Curtis KC, Zhang SR, Li BW, Weil GJ, Rush A

[Filaria J, 2004]

BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.

Knoebel E et al. (2023) MicroPubl Biol "A toolkit for assembly of targeting clones for C. elegans transgenesis."

Dour S, Knoebel E, Nonet M

[MicroPubl Biol, 2023]

Transgenic worms are a key resource for <i>C. elegans</i> researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 <i>SapI</i> GG entry vectors and 100 <i>SapI</i> GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty <i>BsaI</i> -based GG assembly vectors that facilitate the construction of additional <i>SapI</i> GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft&#xae; Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.

Farboud B et al. (2015) Genetics "Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design."

Farboud B, Meyer BJ

[Genetics, 2015]

Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.