Vorremmo ringraziare il Dott. Michael Baker per il suo aiuto nella generazione di costrutti per gene knock-in e per la prestazione del time-lapse film di crescere proiezioni sensoriali. Vorremmo anche ringraziare Virginia Vandelinder di assistenza tecnica con gli esperimenti della pistola pneumatica.

Particelle possono essere rivestiti con diversi reagenti, come le molecole dsRNA, plasmidi DNA o coloranti. Il tipo e il diametro delle particelle sono scelti in base al reagente e la profondità delle cellule bersaglio nei tessuti: le particelle più grandi penetrare ulteriormente, ma può causare qualche danno al tessuto. 1. Preparazione di particelle rivestito dsRNA <ol><li> Posto al 100% isopropanolo sul ghiaccio. </li><li> Risospendere 5 mg di particelle d&#39;oro (S1600ri, Seashell Technology, LLC; diametro medio di 1,6 micron) in 100 ml di tampone vincolanti. Quando la soluzione è pronta, passare alla fase successiva. </li><li> Diluire la soluzione di particelle aggiungendo 100 ml di buffer di legame, mescolare brevemente nel vortex e sonicare per 1-2 minuti per evitare grumi. </li><li> Aggiungi 5-10μg di dsRNA / siRNA (dsRNAs / siRNA deve essere sciolto in acqua ad una concentrazione di circa 1μg/μl). </li><li> Vortex e lasciare a temperatura ambiente per 2 minuti. Questi risultati intervallo di concentrazione e dal tempo in siRNA satura di particelle d&#39;oro. </li><li> Pellet il siRNA / oro complessi delle particelle mediante centrifugazione a ~ 2.500 giri in una centrifuga da banco per ~ 15 sec. </li><li> Rimuovere il surnatante e delicatamente aggiungere 750 ml di isopropanolo freddo con un&#39;interruzione minima del pellet. Spin brevemente e rimuovere il surnatante. </li><li> Risospendere le particelle d&#39;oro in 100 l freddo isopropanolo al 100%, poi sonicare brevemente in un bagno sonicatore (2-3 impulsi) per rompere agglomerati di particelle. Ultimo, versare le particelle in sospensione su un vetrino e lasciare asciugare a temperatura ambiente. </li></ol> 2. Preparare il setup sperimentale per un test specifico <ol><li> Ogni arma ha un diverso diametro di apertura degli ugelli, che vanno da un minimo di 50 micron fino a ~ ~ 3 mm. Le specifiche da utilizzare per la pistola è quindi scelto a seconda della zona di tessuto su cui agire. </li><li> Lui la pressione viene regolata per la profondità nel tessuto delle cellule bersaglio, in genere fino a ~ 100μm (alte pressioni in grado di fornire le particelle più profonda). La distanza tra l&#39;embrione e la punta dell&#39;ugello influisce anche sulla profondità di penetrazione, come momento d&#39;oro particelle libere in aria. </li></ol> 3. Caricamento della pistola con la pre-particelle rivestite Le particelle devono essere caricate come una polvere secca nelle linee di particelle tubi Tygon iniezione collegati al collettore. Questo tubo, che deve essere conservato in un ambiente asciutto a 4 ° C, quando non in uso, possono essere riutilizzati in ulteriori esperimenti con gli stessi reagenti. Il nostro apparato sperimentale in grado di fornire fino a due diversi reagenti per analisi attraverso porte separate nel collettore. <ol><li> Raschiare le particelle essiccate ricoperto dal vetro diapositive utilizzando il bordo di un vetrino di vetro o una lametta prima di caricarli nella linea di tubi Tygon. Questo tubo Tygon saranno specifiche solo per questo reagente per evitare contaminazioni incrociate. </li><li> Come si caricano le particelle, piegare il tubo a forma di U vicino al connettore, per mantenere le particelle di diffondersi lungo il tubo intero e concentrati vicino al connettore. Raccogliere le particelle con una piccola spatola o un pezzo di carta piegato pesano, e caricare circa la metà di loro nel tubo per ogni serie di embrioni. Toccare il tubo durante la fase di carico di diffondere le particelle in modo uniforme. </li></ol> 4. Preparazione degli animali Prima e dopo la consegna delle particelle d&#39;oro, gli embrioni vengono conservati in acqua sterile laghetto artificiale a temperatura ambiente. <ol><li> Mettere gli embrioni, lato ventrale in su, in un solco scavato in un appartamento, 5mm di spessore pezzo di gomma siliconica (Sylgard 184, Dow Corning, Midland, MI) e anestetizzare in una soluzione di 8% di etanolo in acqua sterile laghetto artificiale ( più giovani embrioni in fasi E6-E8 può rimanere in acqua laghetto artificiale senza etanolo tutto l&#39;esperimento). </li><li> Immediatamente prima di sparare le particelle, abbassare il livello bagno per scoprire la superficie ventrale degli embrioni, eliminando una parte della soluzione. Posizionare un pezzo di carta velina con un piccolo foro tagliati a metà su di esso per stabilizzare e mantenere la sua superficie si secchi. </li></ol> 5. Consegna di particelle rivestite in tessuto bersaglio Un carico di particelle in genere può essere utilizzato per un massimo di dieci scatti, con un solo colpo di solito consegna dell&#39;ordine di poche centinaia di particelle. <ol><li> Genera un colpo aprendo una delle valvole di 0,3 s, causando l&#39;iniezione di un bolo di particelle dalla linea di tubazione corrispondente nella pistola. </li><li> Toccare il tubo dolcemente tra gli scatti per rimuovere le particelle dal muro tubi e quindi facilitare la loro iniezione nel flusso di lui la pistola. </li><li> Per coprire l&#39;area di tessuto di interesse, scatti multipli possono essere richiesti. </li><li> Dopo la consegna delle particelle, posto che gli embrioni di nuovo in acqua laghetto artificiale, preferibilmente un embrione per bene in un multi-pozzetti. </li></ol> 6. Immunocolorazione e l&#39;etichettatura dei neuroni Mantenere gli embrioni a temperatura ambiente e al buio per 1 a 3 giorni dopo la consegna delle particelle, fino RNAi (o espressione ectopica) si ottiene. Abbiamo poi eseguire una delle seguenti procedure sui campioni sperimentali e controlli: <ol><li> Ibridazione in situ di test per la presenza o l&#39;assenza di mRNA netrina. Digossigenina marcato sanguisuga riboprobes netrina sono ibridate agli embrioni sanguisuga intero. </li><li> Immunocitochimica al test per le proteine ​​netrina e di altre proteine ​​che sono unici al tessuto neuronale (come anti tubulina acetilata). La procedura viene eseguita anche su tutto il montaggio degli embrioni. </li><li> Microiniezioni di coloranti lipofili (DII e Dio) in singoli neuroni, per la tintura riempie di etichettare i pergolati completa dei neuroni di interesse (neuroni pressione mechanosensory nel nostro caso) in segmenti trattate ei controlli. Questo test viene eseguito su embrioni vivi (intatti o sezionato). </li></ol>

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

In questa dimostrazione, abbiamo usato la pistola pneumatica capillare per fornire particelle d&#39;oro all&#39;interno delle cellule localizzate in piccoli volumi di un embrione vivo sanguisuga per knock-in e knock-down geni. La regione del bersaglio in genere aveva un diametro di circa 150 micron e le particelle sono stati trovati circa 15 micron intorno alla profondità di penetrazione media, che è stato regolabile tra 0 e 50 micron. Quando le particelle sono stati rivestiti con molecole di dsRNA del netrina fattore guida degli assoni, ...

<table><tbody><tr><td>gold particles</td><td>Other</td><td>Seashell Technology, LLC</td><td>S1600ri</td><td></td></tr><tr><td>binding buffer</td><td>Reagent</td><td>Seashell Technology, LLC</td><td></td><td></td></tr><tr><td>silicone rubber </td><td>Reagent</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr></tbody></table>

localized rnai and ectopic gene expression in the medicinal leech

In questo video, che mostrano l&#39;uso di una pistola pneumatica capillare per la consegna accurata biolistic dei reagenti nel tessuto vivo. Usiamo la procedura per i modelli di espressione genica turbare in segmenti selezionati di embrioni sanguisuga, lasciando i segmenti non trattati come controlli interni. La pistola pneumatica capillare può essere utilizzato per raggiungere gli strati interni di cellule nelle fasi precoci dello sviluppo senza aprire il campione. Come metodo per l&#39;introduzione di sostanze localizzato nei tessuti viventi, la consegna biolistic con la pistola ha diversi vantaggi: è veloce, senza contatto e non distruttiva. Inoltre, una pistola singolo capillare può essere utilizzato per la consegna indipendente di sostanze diverse. La regione di consegna può avere dimensioni laterali di circa 50-150 micron e si estende su circa 15 micron intorno alla profondità di penetrazione media, che è regolabile tra 0 e 50 micron. Questa consegna è il vantaggio di essere in grado di indirizzare un numero limitato di celle in una posizione intermedia tra selezionato singola cella abbattere da microiniezione e knockdown sistemico attraverso iniezioni extracellulare o mediante approcci genetici. Per abbattere o bussare nell&#39;espressione del netrina molecola guida degli assoni, che è naturalmente espresso da alcuni neuroni centrali e nella parete ventrale del corpo, ma non il dominio dorsale, forniamo le molecole di dsRNA o plasmide-DNA nella parete del corpo e gangli centrali. Questa procedura prevede le seguenti fasi: (i) la preparazione del setup sperimentale per un test specifico (la regolazione della pressione di accelerazione), (ii) il rivestimento di particelle con le molecole di dsRNA o DNA, (iii) le particelle di carico rivestito nella pistola, fino a due reagenti in un saggio, (iv) preparare gli animali per la consegna delle particelle, (v) la consegna di particelle rivestite in tessuto bersaglio (parete del corpo o gangli), e (vi) il trattamento degli embrioni (immunostaining, immunoistochimica e neuronali etichettatura) per visualizzare i risultati, di solito 2 o 3 giorni dopo il parto. Quando le particelle sono stati rivestiti con netrina dsRNA, hanno causato ben visibili knock-down di espressione netrina che si è verificato solo nelle cellule contenenti particelle (in genere, 1-2 particelle per cella). Particelle rivestite con un plasmide fluorescenza EGFP codifica indotta in cellule neuronali quando si fermarono nel loro nucleo.

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In questo video vi mostriamo una procedura per una consegna precisa biolistic dei reagenti nel tessuto vivo con una pistola romanzo gene in miniatura. Stiamo abbattendo l&#39;espressione della molecola netrina guida degli assoni negli embrioni sanguisuga fornendo molecole di dsRNA nella parete ventrale del corpo e dei gangli di singoli segmenti.

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

localized-rnai-and-ectopic-gene-expression-in-the-medicinal-leech

Research

JoVE Journal

Biology

9.5K Views.  University of California San Diego - UCSD. In questo video vi mostriamo una procedura per una consegna precisa biolistic dei reagenti nel tessuto vivo con una pistola romanzo gene in miniatura. Stiamo abbattendo l'espressione della molecola netrina guida degli assoni negli embrioni sanguisuga fornendo molecole di dsRNA nella parete ventrale del corpo e dei gangli di singoli segmenti.

Video: Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

L&#39;espressione di proteine ​​fluorescenti in<em&gt; Branchiostoma lanceolatum</em&gt; By mRNA iniezione in ovociti non fecondate

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Ricerca

Biologia dello sviluppo

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Il disegno sperimentale per Microdissezione laser RNA-Seq: Lezioni da una analisi dello sviluppo di mais Leaf

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochimica

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Embrione microiniezione e elettroporazione in chordate<em&gt; Intestinalis Ciona</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

Interferenza dell'RNA nei Coleotteri acquatici come potente strumento per manipolare l'espressione genica in punti specifici del tempo di sviluppo

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Ambiente

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

In elettroporazione ovo di miRNA-based plasmidi nel tubo neurale sviluppo e valutazione di fenotipi da Injection DiI in Open-book Preparazioni

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Neuroscienze

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

Sviluppo di ceppi di specie di Leishmania con espressione costitutiva di eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

Genetica

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracellulare di registrazione, sensoriale Campo Mapping, e la coltura neuroni identificata nel Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Biologia

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

Larvali interferenza RNA nella Red Farina Beetle,<em&gt; Tribolium castaneum</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Combinato di nucleotidi e proteina estrazioni in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine fluorescenti in

Il disegno sperimentale per Microdissezione laser RNA-Seq: Lezioni da una analisi dello sviluppo di mais Leaf

Embrione microiniezione e elettroporazione in chordate

Interferenza dell'RNA nei Coleotteri acquatici come potente strumento per manipolare l'espressione genica in punti specifici del tempo di sviluppo

In elettroporazione ovo di miRNA-based plasmidi nel tubo neurale sviluppo e valutazione di fenotipi da Injection DiI in Open-book Preparazioni

Sviluppo di ceppi di specie di Leishmania con espressione costitutiva di eGFP

Intracellulare di registrazione, sensoriale Campo Mapping, e la coltura neuroni identificata nel Leech, Hirudo medicinalis

RNAi di screening per identificare fenotipi postembrionale in C. elegans

Larvali interferenza RNA nella Red Farina Beetle,

Combinato di nucleotidi e proteina estrazioni in Caenorhabditis elegans

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'espressione di proteine ​​fluorescenti in

Il disegno sperimentale per Microdissezione laser RNA-Seq: Lezioni da una analisi dello sviluppo di mais Leaf

Embrione microiniezione e elettroporazione in chordate

Interferenza dell'RNA nei Coleotteri acquatici come potente strumento per manipolare l'espressione genica in punti specifici del tempo di sviluppo

In elettroporazione ovo di miRNA-based plasmidi nel tubo neurale sviluppo e valutazione di fenotipi da Injection DiI in Open-book Preparazioni

Sviluppo di ceppi di specie di Leishmania con espressione costitutiva di eGFP

Intracellulare di registrazione, sensoriale Campo Mapping, e la coltura neuroni identificata nel Leech, Hirudo medicinalis

RNAi di screening per identificare fenotipi postembrionale in C. elegans

Larvali interferenza RNA nella Red Farina Beetle,

Combinato di nucleotidi e proteina estrazioni in Caenorhabditis elegans

L'espressione di proteine fluorescenti in