A First Version of the <i>Caenorhabditis elegans</i> Promoterome

Dupuy, Denis; Li, Qianru; Deplancke, Bart; Boxem, Mike; Hao, Tong; Lamesch, Philippe; Sequerra, Reynaldo; Bosak, Stephanie; Doucette‐Stamm, Lynn; Hope, Ian A.; Hill, David E.; Walhout, Albertha J.M.; Vidal, Marc

doi:10.1101/gr.2497604

Cited by 168 publications

(175 citation statements)

References 48 publications

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“…Davis et al (2008) provide a similar Gateway strategy, but with different Gateway acceptor sites. If the starting sequences are in pDONR221 (Invitrogen), our destination vectors are most useful; if the starting sequences are from the promoterome (Dupuy et al 2004) or ORFeome (Reboul et al 2003) reagents, the Davis et al (2008) Gateway destination vectors may be more convenient.…”

Section: Discussionmentioning

confidence: 99%

“…Additional general information regarding useful strains and constructs: Davis et al (2008) introduce useful constructs that are compatible with promoterome (Dupuy et al 2004) and ORFeome (Reboul et al 2003) reagents; we expand the set of useful FLP-out technology reagents by introducing a variety of additional plasmids for constructing specific FLP recombinase and FLP-out target vectors of choice by both traditional methods and by Gateway cloning methods. The general strategies presented below were used to build plasmids used in this study.…”

Section: Discussionmentioning

confidence: 99%

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A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Voutev

Hubbard

2008

Genetics

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We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.T WO-COMPONENT gene expression systems are indispensable tools to probe molecular mechanisms underlying development. Because control can be exerted by each component independently, exquisite temporal and spatial control of gene activation or repression can be achieved. Using different promoter combinations to drive each component of these systems, additional control can be obtained beyond that afforded by heat-inducible or tissue-specific promoters alone. Site-specific recombination systems such as the FLP/FRT system have been used to control gene expression by ''FLP-out'': a recombinase-catalyzed intramolecular excision of spacer DNA that lies between tandemly oriented FRT sites. The spacer includes a transcriptional stop so that prior to activation of the FLP recombinase (and subsequent FLP-out) the gene downstream of the spacer is not transcribed (Golic and Lindquist 1989;Struhl and Basler 1993; Figure 1A). After the FRT-containing cassette is excised by the FLP recombinase, the downstream gene is brought into proximity to the promoter and is expressed (reporter 2 in Figure 1A). This system and related systems have proven quite powerful and flexible in model organisms including Drosophila and mouse (see Branda and Dymecki 2004, for review;McGuire et al. 2004). However, prior to our study presented here, and a recently published study (Davis et al. 2008), these systems had not been developed for use in Caenorhabditis elegans.An ideal FLP-out system provides the means to generate both loss-and gain-of-function effects in a spatially and temporally controlled manner. In addition, wild-type gene expression can be turned on in particular cells at particular times in an otherwise mutant background. In organisms where transgenes can be reliably inserted in single copy, FLP-out can also be used to eliminate wild-type gene expression by excision of an FRT-flanked wild-type cassette in a mutant background. In C. elegans, the most common methods for generating transgenic C. elegans introduce multiple copies of transgenes on extrachromosomal arrays (Stinchcomb et al. 1985;Mello et al. 1991;Kelly et al. 1997). ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Voutev

Hubbard

2008

Genetics

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“…ptr-6 gDNA was amplified by PCR with C. elegans gDNA as the template. Entry vectors for the promoters were obtained from the Promoterome library (Dupuy et al 2004). For the mboa-1 overexpression experiment, genomic DNA containing 2 kb of the 59 upstream region and the entire coding region of the mboa-1 gene was amplified by PCR.…”

Section: Dna Constructsmentioning

confidence: 99%

Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans

et al. 2016

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Membrane integrity is critical for cell survival, defects of which cause pathological symptoms such as metabolic diseases. In this study, we used ethanol sensitivity of the nematode Caenorhabditis elegans to identify genetic factors involved in membrane integrity. In C. elegans, acute exposure to a high concentration (7% v/v) of ethanol changes membrane permeability, as measured by propidium iodide staining, and causes paralysis. We used the timing of complete paralysis as an indicator for alteration of membrane integrity in our genetic screen, and identified ptr-6 as a gene that confers ethanol resistance when mutated. PTR-6 is a patched-related protein and contains a sterol sensing domain. Inhibition of two PTR-encoding genes, ptr-15 and ptr-23, and mboa-1, encoding an Acyl Co-A: cholesterol acyltransferase homolog, restored ethanol sensitivity of the ptr-6 mutant, suggesting that these ptr genes and mboa-1 are involved in the maintenance of membrane integrity and permeability. Our results suggest that C. elegans can be used as a model system to identify factors involved in metabolic diseases and to screen for therapeutic drugs.KEYWORDS Caenorhabditis elegans; alcohol; Patched-related; membrane integrity P ROPER composition and organization of cellular membranes are important for signaling pathway, cell polarity, and membrane structural stability. Phospholipid composition and thus membrane integrity is often altered in Alzheimer's disease and metabolic diseases such as atherosclerosis and hyperlipidemia (Gillies and Robinson 1988;Muller et al. 1990;Engelmann et al. 1992). Although changes in membrane integrity have been studied intensively in artificial membranes, isolated tissues, and unicellular organisms (Harrison and Vickers 1990;Jung and Levin 1999;Kusumi et al. 2005), the genetic and molecular mechanisms regulating membrane integrity at the multicellular organismal level are largely unknown.Under experimental conditions, membrane integrity can be altered by chemical stresses such as alcohols and detergents. Classical studies found that ethanol influences the fluidity of cell membranes (Chin and Goldstein 1977;Johnson et al. 1979;Dombek and Ingram 1984). Therefore, we reasoned that genetic screens using ethanol as a reagent to alter membrane integrity could lead to isolation of mutants involved in the regulation of membrane integrity. In previous studies, we established a genetic system to identify genes involved in the ethanol response of Caenorhabditis elegans and isolated ethanol-resistant mutants (Kwon et al. 2004;Hong et al. 2008). Realizing that our previous genetic screens might have uncovered mutants involved in the regulation of membrane integrity, we decided to revisit the previously isolated ethanol-resistant mutations. In this study, we analyze the previously isolated ys20 mutant, which we identify as a mutation in the ptr-6 gene. Through further genetic analysis, we propose that cholesterol metabolism is involved in membrane integrity. Our study suggests that the nematode C. elegans ...

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“…As such, the efforts described herein (Dricot et al 2004;Dupuy et al 2004;Lamesch et al 2004;Rual et al 2004a,c), along with other established collections of cDNAs and ORFs (Hudson Jr. et al 1997;Strausberg et al 2002;Carninci et al 2003;Reboul et al 2003) constitute a foundation on which it will be possible to investigate and manipulate both specific genes and proteins and the global networks in which they participate. The creation of multiple types of genome resources, from large-insert genomic DNA libraries to specialized collections of individually cloned genes, cDNAs, and ORFs and their utilization across multiple disciplines as a way to understand biology from a systems approach is a direct consequence of the highly collaborative, interdisciplinary efforts such as those required for the Human Genome Project.…”

Section: From Blueprints To Finished Goodsmentioning

confidence: 99%

A First Version of the Caenorhabditis elegans Promoterome

Cited by 168 publications

References 48 publications

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans

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