M Wayne Davis et al. (2002) West Coast Worm Meeting "Developing new techniques"

Jeff Gritton, M Wayne Davis, Erik M Jorgensen

[West Coast Worm Meeting, 2002]

We have been attempting to add a number of new technologies to the palette of worm genetic techniques. So far, all of these are under development. For the latest updates, or if you have any ideas, please come by my poster. Homologous recombination: The ability to modify chromosomal sequences using homologous recombination would enable targeted knockouts as well as gene replacement. This technique requires the introduction of a linear fragment of DNA, containing free ends, into a germ cell nucleus. Recombination is induced by the free ends, so that the DNA is incorporated into the homologous position on the chromosome. Injection of linear DNA into the syncytial gonad arm never leads to homologous recombination, probably because the free ends are ligated before the DNA gets into a germ cell nucleus. We have tried using I-SceI to liberate a linear fragment from an array (a modification of the Rong and Golic (Science. 2000) approach from flies) with no success. We have also attempted to inject recombinogenic linear fragments directly into the nucleus. These fragments have been ends-in with large 3' overhangs. After a substantial number of injections, we have isolated a number of integrated transgenes, but no homologous events. We also hope to improve the efficiency of recombination by co-injecting the recombination enzyme RAD-51 with our linear DNA. Progress towards this and other approaches will be presented.

M Wayne Davis et al. (2003) International Worm Meeting "A metalloprotease mutant with molting defects"

Aubrey Chan, Erik M Jorgensen, M Wayne Davis

[International Worm Meeting, 2003]

In a number of unrelated screens in our lab we have identified four alleles of a gene with a striking molting defect. After each molt, the partially shed cuticle is dragged behind the worm, held in place by a constriction of the old cuticle around the middle of the worm. We cloned this gene by SNP mapping and sequencing of a candidate metalloprotease, C17G1.6. Sequencing this gene, we found that three alleles have premature stop codons and the fourth allele changes an absolutely conserved histidine residue in the zinc binding site of the enzyme. The gene has an astacin-class zinc metalloprotease domain, an EGF-like domain, a CUB domain and a thrombospondin domain. There are five other closely related genes in the worm genome, including hch-1 and toh-2 (tolloid and BMP-1 related protease 2). hch-1 has similar phenotypes: it is defective for hatching from the egg shell and has defects in migration of QL descendents. These genes are related to hatching enzymes from a number of other organisms, as well as to tolloid proteases. A transcriptional GFP fusion shows that C17G1.6 is expressed in all hypodermal cells in all larval stages. In larval stages before the L4 it appears not to be expressed in the seam cells, but it is strongly expressed in seam cells of the L4. It is possible that the hypodermal expression is increased at larval molts, but further analysis of this expression is required. C17G1.6 is also strongly expressed in the amphid sheath cells, arcade cells, and rectal epithelial cells at all larval stages. It is expressed weakly in the procorpus of the pharynx. In adults, the vulval epithelial cells strongly express C17G1.6, and in older adults these are the only cells expressing GFP. We plan to test C17G1.6 alleles for interaction with hch-1 alleles and RNAi for the other related proteases.

M Wayne Davis et al. (2005) International Worm Meeting "NAS-37 is a conserved metalloprotease that mediates molting"

Andrew J Birnie, Erik M Jorgensen, M Wayne Davis, Aubrey C Chan, Antony P Page

[International Worm Meeting, 2005]

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a C. elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially-shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells four hours prior to ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

M Wayne Davis et al. (2005) International Worm Meeting "High-throughput SNP mapping primers and techniques"

Erik M Jorgensen, Tracey Harrach, Marc Hammarlund, Patrick Hullett, M Wayne Davis, Shawn Olsen

[International Worm Meeting, 2005]

Single nucleotide polymorphism (SNP) mapping against CB4856 has quickly become an indispensable technique in the worm field since its introduction by Stephen Wicks et al. in 2001 (Nat Genet 2001 28: 160-164). Because SNPs are present every few thousand base pairs, SNP mapping allows mapping to extremely narrow intervals. Because mapping is done in an essentially wild-type background, very subtle or complex phenotypes can be mapped that could not be mapped using visible mutations. Typically, SNPs that alter a restriction enzyme recognition site (snip-SNPs) have been detected by running digested PCR products on agarose gels. This technique is reliable and readily accessible to any molecular biology lab; however, it requires substantial hands-on time for each reaction, as each SNP typically requires different PCR cycling and restriction digestion conditions. A number of methods have been published that provide automated analysis of SNP genotypes, including florescence polarization (Swan et al. 2002 Genome Res 12: 1100-1105) or capillary electrophoresis (Zipperlen et al. 2005 Genome Biol 6: R19). Although these methods have the advantage of reducing hands-on time for determining SNP genotypes, they require investment in expensive equipment that is not common in many molecular biology labs. In order to provide a more efficient detection protocol for snip-SNPs, we have developed a set of 48 primer pairs that detect SNPs evenly spaced across the C. elegans genome, and that work under identical PCR and restriction digestion conditions. We present a scheme using these reagents to quickly and reliably map mutations using high-throughput equipment and techniques. We have used our SNP mapping techniques to map many phenotypically subtle or genetically complex mutants, including a subtle behavioral defect resulting from three independent mutations. In fact our techniques are simple and relatively inexpensive enough to have been used successfully by undergraduates in a teaching laboratory setting.

M Wayne Davis et al. (2001) International C. elegans Meeting "Flp-mediated site specific recombination"

M Wayne Davis, Erik M Jorgensen

[International C. elegans Meeting, 2001]

Flp is a site-specific recombinase encoded by the S. cerevisiae 2 micron plasmid. It efficiently catalyzes recombination between two copies of its specific 34 bp recognition site (called the Flp recognition target (FRT)). This reaction works in vitro as well as in vivo in both prokaryotic and eukaryotic cells. In Drosophila, this system is used to generate a variety of recombination events. For example, it has been used to generate tissue specific mosaics by catalyzing mitotic recombination between homologous chromosomes. It has also been used to generate chromosomal deletions, translocations, and inversions. Finally, it has been used as the basis for gene targeting strategies in flies. One major obstacle to the use of the flp/FRT system in C. elegans has been the lack of a simple way to insert single FRT sites into the worm chromosome. I solved this problem by inserting an FRT site into the Mos1 transposon. Germ-line transposition of this element inserts a single, randomly located FRT site into the genome. I currently have 28 insertions of this element in the genome. We have demonstrated that heat-shock driven flp can catalyze recombination in somatic cells between FRT elements on a transgene. In order to express flp in the germline, I have made transgenic arrays containing complex DNA. I now plan to use these reagents to test the feasibility of using flp/FRT to generate heritable rearrangements in worms. We are exploring several possible applications of the flp/FRT system in C. elegans including: 1. Generation of a library of small, molecularly defined chromosomal deletions. These deletions would be extremely useful in genetic mapping. Further, these could be screened for haploinsufficient genes, or for early lethal phenotypes when homozygous. 2. Generation of new balancers. Currently most balancers are translocations; only a few inversion balancer chromosomes presently exist. Flp/FRT can be used to generate inverted chromosomes for balancers. 3. Mitotic chromosome segregation. C. elegans has holocentric chromosomes. This means that centromere function is distributed along the entire length of the chromosome. We will use flp-mediated recombination to explore how holocentric chromosomes segregate after the induction of mitotic recombination. 4. Tissue- and time-dependent transgene control. If a rescuing transgene is generated such that part of the ORF is flanked by tandem FRT sites, the ORF can be specifically disrupted by expression of the flp recombinase. The recombinase can be expressed either by tissue-specific or heat-induced promoters to eliminate gene function in a specific cell type or at a specific time. Conversely, FRT containing constructs can be designed so that expression of a gene can be induced by flp expression. 5. Inserting single copy transgenes. flp/FRT can catalyze the transfer of a piece of DNA that is flanked by tandem FRT sites into a single FRT site on another molecule. This reaction could be used to move a region of a transgene into an FRT located on a chromosome, generating a single copy transgene. This approach has the added benefit that different DNA sequences could be individually recombined into the same chromosomal FRT site, allowing for identical chromosomal environment of different insertions. 6. If you have any other ideas please drop by my poster.

M. Wayne Davis et al. (2007) International Worm Meeting "Conditional knockouts using TEV protease expression."

Erik M. Jorgensen, Chris Hopkins, M. Wayne Davis, Christian Frokjaer-Jensen

[International Worm Meeting, 2007]

Deletion alleles and RNAi knock-down are able to show the null phenotype for a gene of interest. However, if the null allele is lethal it can be impossible to analyze the function of the gene in the adult. Often what is needed is a way to induce the acute disruption of the gene product at a specific time or in specific cells. We are attempting to generate acute disruption using the site-specific TEV protease. TEV is a viral protease with a 7 amino acid recognition sequence. Because of its high sequence specificity, TEV protease can be expressed in vivo without leading to detectable effects on the cell. By engineering a TEV cleavage site into the protein of interest, TEV protease expression has been used to acutely disrupt protein function in both bacteria and yeast(1, 2). We built a construct that expresses the TEV protease under the hsp-16.41 heat-shock promoter. As in other systems, we found no obvious deleterious effects when transgenic animals are heat-shocked. Native TEV protease contains an internal cleavage site, and so is self-inactivating; however, we are using the S219V mutation that is resistant to this inactivation(3). As a test for TEV cleavage of a substrate in C. elegans, we designed a fusion between GFP and clathrin heavy chain (CHC-1) with a TEV cleavage site between them. This fusion protein was put under the control of the GABA neuron promoter Punc-47. Before TEV induction, GFP is localized to GABA synapses, consistent with known CHC-1 distribution. After TEV induction, GFP becomes diffuse in the cell, suggesting that it is no longer attached to clathrin. This result is confirmed by Western blot. To determine the time course of TEV cleavage in vivo, we built a CFP-YFP fusion protein joined by a TEV cleavage site. TEV cleavage of this protein can be detected by a drop in the fluorescence resonance energy transfer from CFP to YFP. We found that the protein cleavage is maximal about two hours after the start of a one hour heat shock. We are now testing whether TEV cleavage of an engineered protein of interest can recapitulate its null phenotype by introducing a TEV cleavage site into the synaptic protein UNC-18. 1.M. Mondigler, M. Ehrmann, J Bacteriol 178, 2986 (May, 1996). 2.X. Yang, J. Gregan, K. Lindner, H. Young, S. E. Kearsey, BMC Mol Biol 6, 13 (2005). 3.R. B. Kapust et al., Protein Eng 14, 993 (Dec, 2001).

M. Wayne Davis et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Conditional knockouts using TEV protease expression"

M. Wayne Davis, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Deletion alleles and RNAi knock-down are able to show the null phenotype for a gene of interest. However, if the null allele is lethal it is impossible to analyze the function of the gene in the adult. Often what is needed is a way to induce the acute disruption of the gene product at a specific time or in specific cells. We are attempting to generate acute disruption using the site-specific TEV protease. TEV is a viral protease with a seven amino acid recognition sequence. Because of its tight sequence specificity, it has been shown that TEV protease can be expressed in vivo without leading to detectable effects on the cell, and TEV protease expression has been used to acutely disrupt protein function in both bacteria and yeast(1, 2). Native TEV protease contains an internal cleavage site, and so is self-inactivating; however, the S219V mutation is resistant to this inactivation(3). We have generated a construct that expresses the TEV protease S219V mutant (from D.S. Waugh) under the hsp-16.41 heat-shock promoter and have found no obvious deleterious effects when transgenic animals are heat-shocked. As a test for TEV cleavage of a substrate in vivo, we have generated a fusion between GFP and clathrin heavy chain (CHC-1) with a TEV cleavage site between them. This fusion protein was then put under the control of the GABA neuron promoter Punc-47. Before TEV induction, GFP is localized to GABA synapses. After induction, GFP appears to be more diffuse in the cell, suggesting that it is no longer attached to clathrin. We are currently quantitating cleavage efficiency using western blots.

Rich, Matthew S. et al. (2019) International Worm Meeting "Characterizing neuronal gene expression with a 'Standard Worm."

Jorgensen, Erik, Rich, Matthew S., Davis, M. Wayne

[International Worm Meeting, 2019]

An adult C. elegans has only 959 cells, 302 of which are neurons. Due to the efforts of the last few decades, we know the synaptic connections of every neuron and the major functions of most. Because C. elegans develops along a fixed cell lineage, every neuron can be uniquely identified - this is a difficult, time-consuming, and error prone process, though. We require new tools that can automate the neuron and cell identification process in C. elegans, making it simpler, faster, and more reproducible. To fill this need, we are developing the "Standard Worm," an C. elegans strain in which all neurons are labeled by a deterministic combination of fluorescent proteins. The current iteration of the Standard Worm uses a set of four colors and seven promoters that labels nearly all neurons in the animal, leaving citrine (YFP) as a low-background reporter for gene expression. We are also developing software to automatically segment images and annotate neurons, using both the color combinations and known positions of nuclei to aid analysis. With the advent of high-throughput genome engineering technologies, we will soon make large, genome-scale collections of gene knockouts and transcriptional reporters. Already, researchers are using calcium indicators to measure neuron activity in all C. elegans neurons. It is our hope that the Standard Worm will enable simple single-neuron analysis of these datasets, leading to a more complete understanding of the development and function of the C. elegans nervous system.

Schwartz, Matthew et al. (2019) International Worm Meeting "Efficient Cre/Lox recombinase-mediated cassette exchange in C. elegans."

Rich, Matthew S., Jorgensen, Erik, Schwartz, Matthew, Davis, M. Wayne

[International Worm Meeting, 2019]

Recombinase-mediated cassette exchange is a well established method to insert genetic material into a genome at predefined landing sites. Briefly, individual DNA sequences flanked by recombinase sites can be inserted into a genomic locus containing matching recombinase sites. To date, efficient cassette exchange protocols have not been developed for C. elegans. Employing a genomically integrated, germline expressed Cre transgene, we have recently achieved efficient Cre/Lox cassette exchange in C. elegans. In initial experiments, >50% of P0's injected with ~5 kb exchange cassettes yield at least one cassette exchange event amongst their progeny. We are developing a suite of worm strains, plasmids and protocols to leverage cassette exchange for a variety of genome editing operations, including inserting entire transgenes at pre-defined safe-harbor loci and 'swapping' tags initially inserted into native genes using CRISPR. Ultimately, we seek to develop cassette exchange into a robust, multipurpose tool to complement CRISPR in the worm engineer's toolkit.

Davis RE (1996) Parasitol Today "Spliced leader RNA trans-splicing in metazoa."

Davis RE

[Parasitol Today, 1996]

Spliced leader trans-splicing is a form of RNA processing originally described and studied in parasitic kinetoplastida. This mechanism of gene expression also occurs in parasitic and free-living metazoa. In this review, Dick Davis describes current knowledge of the distribution, substrates, specificity and functional significance of trans-splicing in metazoa.