Ringraziamo: Nicolas Gompel e Benjamin Prud&#39;homme per il loro contributo allo sviluppo di questo protocollo, Melissa Williams e quattro revisori anonimi per i commenti sul manoscritto, l&#39;Università di Dayton Scuola di specializzazione per borse di ricerca in WAR, e la University of Dayton Biologia Dipartimento e Research Institute (UDRI) per sostenere la ricerca per TMW. Questo lavoro è stato sostenuto dalla American Heart Association Concessione 11BGIA7280000 a TMW.

<ol>
	<li>Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Regulatory+Genome.">The Regulatory Genome.</a> Gene Regulatory Networks in Development and Evolution. , (2006).</li><li>Wray, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+significance+of+cis-regulatory+mutations.">The evolutionary significance of cis-regulatory mutations.</a> Nat. Rev. Genet. 8, 206-216 (2007).</li><li>Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+developmental+biology+and+the+problem+of+variation.">Evolutionary developmental biology and the problem of variation.</a> Evolution. 54, 1079-1091 (2000).</li><li>Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo+and+an+expanding+evolutionary+synthesis:+a+genetic+theory+of+morphological+evolution.">Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.</a> Cell. 134, 25-36 (2008).</li><li>Sethupathy, P., Collins, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+target+site+polymorphisms+and+human+disease.">MicroRNA target site polymorphisms and human disease.</a> Trends Genet. 24, 489-497 (2008).</li><li>Visel, A., Rubin, E. M., Pennacchio, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+views+of+distant-acting+enhancers.">Genomic views of distant-acting enhancers.</a> Nature. 461, 199-205 (2009).</li><li>Spradling, A. C., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+cloned+P+elements+into+Drosophila+germ+line+chromosomes.">Transposition of cloned P elements into Drosophila germ line chromosomes.</a> Science. 218, 341-347 (1982).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 3312-3317 (2007).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. , (2008).</li><li>Rebeiz, M., Pool, J. E., Kassner, V. A., Aquadro, C. F., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+modification+of+a+modular+enhancer+underlies+adaptation+in+a+Drosophila+population.">Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population.</a> Science. 326, 1663-1667 (2009).</li><li>Williams, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+evolution+of+a+genetic+switch+controlling+sexually+dimorphic+traits+in+Drosophila.">The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.</a> Cell. 134, 610-623 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS biology. 7, e1000168-e1000168 (2009).</li><li>Barolo, S., Carver, L. A., Posakony, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+and+beta-galactosidase+transformation+vectors+for+promoter/enhancer+analysis+in+Drosophila.">GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila.</a> BioTechniques. 29, 726-732 (2000).</li><li>Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid. , (2007).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. , (2005).</li><li>Abramoff, M. D., Magelhaes, P. S., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Musunuru, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+noncoding+variant+to+phenotype+via+SORT1+at+the+1p13+cholesterol+locus.">From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus.</a> Nature. 466, 714-721 (2010).</li></ol>

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cis-normativo elementi di codificare il programma genomico che specifica gli schemi di espressione genica e quindi il processo di sviluppo 1, e sono luoghi importanti per entrambe le mutazioni alla base dell&#39;evoluzione morfologica 2-4 e variazione fenotipica di tratti umani 5,6,19. A dispetto di questa importanza, la logica di regolamentazione per CRES sono ancora limitate. Una ragione importante per comprendere questo deficit è stata la mancanza di metodi adeguati per conf...

quantitative comparison of cis-regulatory element (cre) activities in transgenic drosophila melanogaster

Schemi di espressione genica sono specificati dal cis-regolamentazione elemento (CRE) sequenze, che sono anche chiamati esaltatori o cis-regolamentazione moduli. Un tipico CRE possiede un accordo di siti di legame per il fattore di trascrizione diverse proteine ​​che conferiscono una logica regolamentare specificando quando, dove ea quale livello del gene regolamentati (s) è espresso. Il set completo di CRES all&#39;interno di un programma di codifica del genoma animale dell&#39;organismo per lo sviluppo 1, ed empirica come pure studi teorici indicano che mutazioni a Cres svolto un ruolo preminente nella evoluzione morfologica 2-4. Inoltre, gli studi del genoma umano gamma di associazione indicano che la variazione genetica a Cres contribuire in modo sostanziale alla variazione fenotipica 5,6. Così, la comprensione logica regolamentare e di come le mutazioni influenzano tale logica è un obiettivo centrale della genetica. Transgeni reporter di fornire un metodo efficace per studiare in vivo la funzionezione di Cres. Ecco una sequenza di note o sospette CRE è accoppiato ad promotore eterologo e sequenze che codificano per un gene reporter codifica per una proteina prodotta facilmente osservabili. Quando un giornalista transgene si inserisce in un organismo ospite, l&#39;attività del CRE diventa visibile sotto forma della proteina codificata giornalista. P-elemento transgenesi mediata nella specie di moscerino della frutta Drosophila (D.) melanogaster 7 è stato utilizzato per decenni per introdurre transgeni giornalista in questo organismo modello, anche se la collocazione genomica dei transgeni è casuale. Quindi, l&#39;attività del gene reporter è fortemente influenzato dalla cromatina locale e l&#39;ambiente gene, limitando confronti CRE di essere qualitativa. Negli ultimi anni, il sistema di integrazione basato phiC31 è stato adattato per l&#39;uso in D. melanogaster per inserire transgeni in specifici siti del genoma di atterraggio 10/08. Questa capacità ha reso la misurazione quantitativa del gene e, qui interessa, l&#39;attività CRE 11-13 feasible. La produzione di moscerini della frutta transgenica possono essere esternalizzate, tra cui phiC31 a base di integrazione, eliminando la necessità di acquistare costose attrezzature e / o competenza specialistica a protocolli di iniezione transgene. Qui vi presentiamo un protocollo generale per valutare quantitativamente un CRE l&#39;attività, e mostrare come questo approccio può essere utilizzato per misurare gli effetti di una mutazione introdotta l&#39;attività di un CRE e confrontare le attività del CRES ortologhi. Anche se gli esempi riportati sono per un attivo CRE durante metamorfosi moscerino della frutta, l&#39;approccio può essere applicato ad altre fasi di sviluppo, specie di moscerino della frutta, o di organismi modello. In definitiva, un uso più diffuso di questo approccio a Cres studio dovrebbe avanzare la comprensione della logica normativa e come logica può variare ed evolvere.

Watch this Scientific Journal Video about Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster at JoVE.com

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Variazione fenotipica per i tratti può derivare da mutazioni in cis-regolamentazione elemento (CRE) sequenze che i modelli di espressione genica di controllo. Metodi derivati ​​per l&#39;utilizzo in Drosophila melanogaster possono confrontare quantitativamente i livelli di modelli spaziali e temporali di espressione genica mediata da modificato o naturale varianti CRE.

Confronto quantitativo di Cis-Regulatory Element (CRE) Attività in transgenici Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

quantitative-comparison-cis-regulatory-element-cre-activities

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

11.7K Views.  University of Dayton. Variazione fenotipica per i tratti può derivare da mutazioni in cis-regolamentazione elemento (CRE) sequenze che i modelli di espressione genica di controllo. Metodi derivati ​​per l'utilizzo in Drosophila melanogaster possono confrontare quantitativamente i livelli di modelli spaziali e temporali di espressione genica mediata da modificato o naturale varianti CRE.

Video: Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

Multi-bersaglio Cromogenico Whole-mount<em&gt; In Situ</em&gt; Ibridazione per il confronto Gene domini di espressione in<em&gt; Drosophila</em&gt; Embrioni

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Ricerca

Biologia dello sviluppo

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificare le attività di Cis-Regolamentazione Elementi nella retina di topo Elettroporazione Espianto

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Neuroscienze

In-Nucleus Hi-C in Drosophila Cells

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

The genome is organized in the nuclear space into different structures that can be revealed through chromosome conformation capture technologies. The in-nucleus Hi-C method provides a genome-wide collection of chromatin interactions in Drosophila cell lines, which generates contact maps that can be explored at megabase resolution at restriction fragment level.

In-Nucleus Hi-C nelle cellule di Drosophila

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, ...

Genetica

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Cella di aggregazione saggi per valutare l'associazione della tacca della drosofila con Trans-ligandi e la sua inibizione di Cis-ligandi

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

phiC31 Transgenesi embrionale mediata dall'integrasi in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Autore in primo piano: Ottimizzazione della microiniezione di embrioni per la transgenesi in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Microiniezione di embrioni per la transgenesi in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Sistemica infezione batterica e immunitario Difesa fenotipi in<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Immunologia e infezioni

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Determinazione del locomotore attività spontanea in<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Confronto quantitativo di Cis-Regulatory Element (CRE) Attività in transgenici Drosophila melanogaster

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Multi-bersaglio Cromogenico Whole-mount

Quantificare le attività di Cis-Regolamentazione Elementi nella retina di topo Elettroporazione Espianto

In-Nucleus Hi-C nelle cellule di Drosophila

Cella di aggregazione saggi per valutare l'associazione della tacca della drosofila con Trans-ligandi e la sua inibizione di Cis-ligandi

Microiniezione di embrioni per la transgenesi in Drosophila

Microiniezione di embrioni per la transgenesi in Drosophila

Microiniezione di embrioni per la transgenesi in Drosophila

Sistemica infezione batterica e immunitario Difesa fenotipi in

Determinazione del locomotore attività spontanea in

Microdissezione laser applicata a profili di espressione genica di sottogruppo di cellule dal Drosophila Ala Disco