Rapid selection of transgenic C. elegans using antibiotic resistance

Semple, Jennifer I.; Garcia-Verdugo, Rosa; Lehner, Ben

doi:10.1038/nmeth.1495

Cited by 77 publications

(87 citation statements)

References 12 publications

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“…As with other metazoans, nematodes are sensitive to broadspectrum antibiotics, such as G-418, puromycin, and phleomycin [72,73]. G-418 and puromycin interfere with the function of ribosomes and block protein synthesis in eukaryotes and prokaryotes [51,74], while phleomycin inhibits DNA synthesis, again both in eukaryotes and prokaryotes [75].…”

Section: Antibiotic Markersmentioning

confidence: 99%

“…G-418 and puromycin interfere with the function of ribosomes and block protein synthesis in eukaryotes and prokaryotes [51,74], while phleomycin inhibits DNA synthesis, again both in eukaryotes and prokaryotes [75]. G-418 has been shown to be toxic to C. elegans in liquid and solid culture at concentrations higher than 0.1 mg/ml [72,73]. As had already been shown in Drosophila [67], hatchlings are more sensitive to the drug than young adults, probably due to a higher dependency on their protein synthesis machinery for development.…”

Section: Antibiotic Markersmentioning

confidence: 99%

“…Accordingly, Giordano-Santini and co-workers defined a critical concentration of G-418 for solid culture (0.4 mg/ml), at which L1 larvae are unable to develop and die after a few days while young adults are unaffected and are able to lay eggs. Puromycin is also toxic to C. elegans both in liquid and solid culture, at concentrations similar to that of G-418 [73]. Finally, the sensitivity of C. elegans to phleomycin has so far only been shown in liquid culture and is relatively low, presumably because cells in intact worms are relatively inaccessible to the drug [73].…”

Section: Antibiotic Markersmentioning

confidence: 99%

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Selectable genetic markers for nematode transgenesis

Giordano-Santini

Dupuy

2011

Cell. Mol. Life Sci.

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The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.

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Section: Antibiotic Markersmentioning

confidence: 99%

Section: Antibiotic Markersmentioning

confidence: 99%

Section: Antibiotic Markersmentioning

confidence: 99%

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Selectable genetic markers for nematode transgenesis

Giordano-Santini

Dupuy

2011

Cell. Mol. Life Sci.

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“…For this reason, a site-specific recombination-based DNA cloning method was developed that circumvents the use of restriction enzymes (Hartley et al, 2000). The advent of recombination-based cloning brought a series of diverse and pioneering studies showing the utility of this technology in creating DNA constructions for transgenesis (Fisher et al, 2006;Hope et al, 2004;Ikeya et al, 2005;Kappas et al, 2008;Kwan et al, 2007;Nyabi et al, 2009;Semple et al, 2010;Skarnes et al, 2011). However, none had yet been designed specifically for use in Xenopus, a widely used model organism (Amaya, 2005), and the ability to use them across multiple models was limited.…”

Section: Introductionmentioning

confidence: 99%

pTransgenesis: a cross-species, modular transgenesis resource

et al. 2011

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SUMMARYAs studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.

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“…For example, individual transgenic worms are picked on the basis of a visible selection marker, such as GFP. As for alternative approaches, difficulty in obtaining large transgenic populations can be often circumvented by coinjecting a rescuing transgene in a variety of morphological or lethal mutant backgrounds, or by coinjecting an antibiotic resistance gene (14,15). The behavioral assays should be combined with one of these selection procedures.…”

mentioning

confidence: 99%

High-throughput optical quantification of mechanosensory habituation reveals neurons encoding memory in Caenorhabditis elegans

Sugi

Ohtani

Kumiya

et al. 2014

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

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A major goal of neuroscience studies is to identify the neurons and molecules responsible for memory. Mechanosensory habituation in Caenorhabditis elegans is a simple form of learning and memory, in which a circuit of several sensory neurons and interneurons governs behavior. However, despite the usefulness of this paradigm, there are hardly any systems for rapid and accurate behavioral genetic analysis. Here, we developed a multiplexed optical system to genetically analyze C. elegans mechanosensory habituation, and identified two interneurons involved in memory formation. The system automatically trains large populations of animals and simultaneously quantifies the behaviors of various strains by optically discriminating between transgenic and nontransgenic animals. Biochemical and cell-specific behavioral analyses indicated that phosphorylation of cyclic AMP response element-binding protein (CREB), a factor known to regulate memory allocation, was facilitated during training and this phosphorylation in AVA and AVD interneurons was required for habituation. These interneurons are a potential target for cell-specific exploration of the molecular substrates of memory.Caenorhabditis elegans | mechanosensory habituation | memory | optical quantification | neural circuit I dentification of the physical substrates of memory in the brain, namely neurons and molecules encoding memory, is a major challenge in neuroscience. Recent studies have revealed that the transcription factor cAMP response element-binding protein (CREB) (1) is crucial for determining which neurons in the lateral amygdala participate in encoding an auditory fear memory, and that neurons with higher CREB activity are preferentially allocated into a unique memory trace (memory engram) (2-6). Thus, it has been generally thought through the accumulated weight of evidence that memory is represented by a specific population of neurons in the brain that forms memory engrams: only a portion of eligible neurons are recruited into a specific memory. Although this CREB-dependent memory allocation model is established (5), the identification of specific neurons supporting a given memory remains a long-standing challenge. Moreover, the extreme complexity of nervous systems in model animals still hampers cell-specific genetic analyses; therefore, even if neurons encoding memory could be identified, it would be difficult to address the subsequent question of how these neurons store memory at the molecular level in situ.The nematode Caenorhabditis elegans is a genetically tractable model organism with a neural circuit composed of only 302 neurons. Moreover, the wiring of this nervous system has been completely determined. These unique features of the C. elegans nervous system have proven to be advantageous for various behavioral experiments, such as genetic rescue experiments, ectopic expression analysis, overexpression analysis, and RNA interference-mediated gene disruption, because all of these transgenic studies can be conducted in situ in a cell-specific manner...

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Rapid selection of transgenic C. elegans using antibiotic resistance

Cited by 77 publications

References 12 publications

Selectable genetic markers for nematode transgenesis

Selectable genetic markers for nematode transgenesis

pTransgenesis: a cross-species, modular transgenesis resource

High-throughput optical quantification of mechanosensory habituation reveals neurons encoding memory in Caenorhabditis elegans

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