Single-copy insertion of transgenes in Caenorhabditis elegans

Frøkjær-Jensen, Christian; Davis, Matthew L.; Hopkins, Christopher E.; Newman, Blake J.; Thummel, Jason M.; Olesen, Søren‐Peter; Grunnet, Morten; Jørgensen, Erik M.

doi:10.1038/ng.248

Cited by 1,130 publications

(1,349 citation statements)

References 26 publications

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“…We generated transgenic worm strains that produced MSP-142 labeled on the N terminus with tagRFP-T (7) specifically in sperm cells via either the peel-1 promotor (8) or the spe-11 promotor, using the Mos1-mediated single-copy insertion technique (9) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Membrane tension regulates motility by controlling lamellipodium organization

Batchelder

Hollopeter

Campillo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

136

114

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Many cell movements proceed via a crawling mechanism, where polymerization of the cytoskeletal protein actin pushes out the leading edge membrane. In this model, membrane tension has been seen as an impediment to filament growth and cell motility. Here we use a simple model of cell motility, the Caenorhabditis elegans sperm cell, to test how membrane tension affects movement and cytoskeleton dynamics. To enable these analyses, we create transgenic worm strains carrying sperm with a fluorescently labeled cytoskeleton. Via osmotic shock and deoxycholate treatments, we relax or tense the cell membrane and quantify apparent membrane tension changes by the membrane tether technique. Surprisingly, we find that membrane tension reduction is correlated with a decrease in cell displacement speed, whereas an increase in membrane tension enhances motility. We further demonstrate that apparent polymerization rates follow the same trends. We observe that membrane tension reduction leads to an unorganized, rough lamellipodium, composed of short filaments angled away from the direction of movement. On the other hand, an increase in tension reduces lateral membrane protrusions in the lamellipodium, and filaments are longer and more oriented toward the direction of movement. Overall we propose that membrane tension optimizes motility by streamlining polymerization in the direction of movement, thus adding a layer of complexity to our current understanding of how membrane tension enters into the motility equation. I n crawling cells, motility is mainly driven by actin polymerization, which forms filaments beneath the leading edge cell membrane to make protrusions (1). In this scenario, polymerization is inhibited by the presence of the cell membrane, and membrane tension is thus commonly seen as an impediment to cell motility (2). Experiments show that lamellipodial extension rate is indeed inversely correlated with membrane tension (3), but steady-state, whole cell translocation has not been studied. Here we use a simplified system of crawling cell motility, the Caenorhabditis elegans sperm cell, in order to address the question of how membrane tension affects whole cell translocation. The sperm cell contains only cytoskeleton, mitochondria, and nuclear material and is incapable of de novo protein synthesis or classical exo-and endocytosis, thus representing a useful model for exploring the interplay between membrane tension and cell motility. The sperm cell translocates by adhering to the substrate and emitting a dynamic lamellipodia, in a manner that is morphologically similar to actomyosin containing motile cells, despite the fact that the movement of sperm cells is powered by the dynamics of the major sperm protein (MSP) cytoskeleton in the absence of actin and known molecular motors (4, 5).The production of fluorescently labeled actin in living cells was a watershed in the understanding of how actin structures assemble, flow, and disassemble to produce cell motility. However, due to efficient gene silencing mechanisms in th...

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Section: Resultsmentioning

confidence: 99%

Membrane tension regulates motility by controlling lamellipodium organization

Batchelder

Hollopeter

Campillo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

136

114

View full text Add to dashboard Cite

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“…We know little about CEP-1 post-translational modi fi cations. It will be important to identify the modi fi cations and to determine their importance in knock-in experiments, which should be possible using transposon excision-based gene replacement technologies (Frokjaer-Jensen et al 2008 ) .…”

Section: Cep-1 Regulationmentioning

confidence: 99%

Germ Cell Apoptosis and DNA Damage Responses

Bailly

Gartner

2012

Advances in Experimental Medicine and Biology

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In the past 12 years, since the fi rst description of C. elegans germ cell apoptosis, this area of research rapidly expanded. It became evident that multiple genetic pathways lead to the apoptotic demise of germ cells. We are only beginning to understand how these pathways that all require the CED-9/Bcl-2, Apaf-1/CED-4 and CED-3 caspase core apoptosis components are regulated. Physiological apoptosis, which likely accounts for the elimination of more than 50% of all germ cells, even in unperturbed conditions, is likely to be required to maintain tissue homeostasis. The best-studied pathways lead to DNA damage-induced germ cell apoptosis in response to a variety of genotoxic stimuli. This apoptosis appears to be regulated similar to DNA damage-induced apoptosis in the mouse germ line and converges on p53 family transcription factors. DNA damage response pathways not only lead to apoptosis induction, but also directly affect DNA repair, and a transient cell cycle arrest of mitotic germ cells. Finally, distinct pathways activate germ cell apoptosis in response to defects in meiotic recombination and meiotic chromosome pairing.

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“…Maps for these reporters are available upon request. Injection marker plasmids pGH8 and pCFJ104 (used for lfIs177) are described in the study by Frøkjaer-Jensen et al 42 Immunostainings and RAD-51 foci quantification. Germlines were dissected from young adults (1 day post-L4 stage) and processed for immunostaining 43 .…”

Section: Methodsmentioning

confidence: 99%

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

et al. 2014

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Genomes contain many sequences that are intrinsically difficult to replicate. Tracts of tandem guanines, for instance, have the potential to adopt stable G-quadruplex structures, which are prone to cause genome alterations. Here we describe G4 DNA-induced mutagenesis in Caenorhabditis elegans and identify a non-canonical DNA break repair mechanism that generates deletions characterized by an extremely narrow size distribution, minimal homology of exactly one nucleotide at the junctions, and by the occasional presence of templated insertions. This typical mutation profile is fully dependent on the A-family polymerase Theta, the absence of which leads to profound loss of sequences surrounding G4 motifs. Theta-mediated end-joining prevails over non-homologous end joining and homologous recombination and prevents genomic havoc at replication fork barriers at the expense of small deletions. G4 DNA-induced deletions also manifest in the genomes of wild isolates of C. elegans, indicating a protective role for this pathway during evolution.

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Single-copy insertion of transgenes in Caenorhabditis elegans

Cited by 1,130 publications

References 26 publications

Membrane tension regulates motility by controlling lamellipodium organization

Membrane tension regulates motility by controlling lamellipodium organization

Germ Cell Apoptosis and DNA Damage Responses

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

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