Questo lavoro è stato sostenuto dalle concessioni di AK dalla Israel Science Foundation, il ministero israeliano della sanità, e DFG (tedesco Research Foundation).

I. Elettroporazione Uovo di movimentazione <ol><li> Mettere le uova in un set umidificata incubatore a 37-38 ° C, preferibile un incubatore con vassoi a dondolo. </li><li> Embrione sono elettroporate dopo circa 66 ore di incubazione, quando hanno raggiunto Hamburger &amp; Hamilton (HH) stadio 18-20. In questa fase la testa si trova ad angolo retto rispetto al tronco (condizione chiamata cervicale flessione) e una vasta serie di extra-embrionali vasi sanguigni, come anteriore, posteriore, destro e sinistro vene vitellina dovrebbe essere visto. Questa è una fase ottimale per enhancer-dipendente specifica espressione 1,3,4. E &#39;importante monitorare temperatura e umidità dell&#39;incubatore, al fine di ottenere embrioni vitali e sincronizzati. </li><li> Dopo 66 ore di incubazione, togliere le uova dal termostato. Mettere le uova in orizzontale. Attendere 5-10 minuti. L&#39;embrione ora si trova al polo superiore dell&#39;uovo. </li></ol> Preparativi <ol><li> Preparare la soluzione di Hank con penicillina / streptomicina (P / S) (diluizione 1:100). </li><li> Tirare in vetro capillari (0,5 mm di diametro) a microcapillare. </li><li> Posizionare gli elettrodi (tungsteno) in micromanipolatore e collegare gli elettrodi al generatore di impulsi (ECM 830). </li><li> Regolare il generatore di impulsi con i seguenti parametri: 30v tensione, numero di impulsi - 3, lunghezza dell&#39;impulso di 50 ms. </li><li> Preparare una pipetta a bocca, un ago della siringa e di un nastro. </li><li> Preparare una miscela desiderata di DNA (2-5μg/μl) e aggiungere un colorante Fast Green. </li></ol> Finestre ed elettroporazione <ol><li> Utilizzando una siringa, togliere 5-6 ml di albumina da ogni uovo. </li><li> Con una piccola forbice aperta una finestra ovale sul lato superiore dell&#39;uovo. </li><li> Umida l&#39;embrione con 0,5-1ml di Hank + P / S soluzione. </li><li> Utilizzare una pipetta a bocca, per caricare il vetro microcapillare con la miscela di DNA. </li><li> Posto l&#39;embrione con la coda verso di voi. In questa fase, il midollo spinale degli embrioni è chiaramente visibile. Pertanto, non sono richieste tecniche di contrasto. Il midollo spinale è sigillato alle due estremità, la testa e la coda, ma se il tuo microcapillare è abbastanza sottile, si sarà in grado di perforare un piccolo buco e penetrare il tubo neurale con un angolo basso. Un microcapillare di diametro diritto devono essere caricati facilmente con il DNA, penetrare il tubo senza danneggiarlo e rilasciare il DNA con una espirazione normale. </li><li> Iniettare il DNA usando una pipetta a bocca. Il colorante verde dovrebbe diffondersi dalla punta della coda fino alle vescicole di sviluppo del cervello. </li><li> Posizionare gli elettrodi in parallelo al tubo neurale, il più vicino possibile, senza toccare il tubo stesso e impulsi. Al momento dell&#39;impulso, gli elettrodi devono essere coperti con il liquido per consentire la conducibilità elettrica. Di solito, la quantità di Hank aggiunto a chiedere l&#39;elemosina è sufficiente. Se non aggiungere una goccia di Hank appena prima del polso. </li><li> Rimuovere con attenzione gli elettrodi e sigillare la finestra con un nastro. E &#39;importante per sigillare l&#39;uovo interamente contro l&#39;essiccamento della dell&#39;embrione durante il periodo di incubazione successivo. </li><li> Posizionare la parte posteriore uova nell&#39;incubatrice. </li></ol> II. Midollo spinale libro aperto preparazione Distacco del midollo spinale dall&#39;embrione <ol><li> Rimuovere l&#39;embrione eletroporated, a E6, da l&#39;uovo e metterlo in una capsula di Petri rivestito con silicone contenente PBS. </li><li> Tagliare le membrane e allungare l&#39;embrione dalla sua parte ventrale mediante perni per tenerlo. </li><li> Utilizzando un tungsteno nitide microcapillare fare una incisione longitudinale lungo la lamiera del tetto, dal romboencefalo fino alla coda. </li><li> Fare un ulteriore due incisioni longitudinali su entrambi i lati del midollo spinale, staccando i gangli della radice dorsale (DRG) dal midollo spinale. </li><li> Staccare la piastra dal tessuto partendo dalla coda al romboencefalo, lasciando intatto il midollo spinale. </li><li> Tagliare il midollo spinale in una sezione trasversale al romboencefalo con le forbici fine e separato dal corpo. </li></ol> Fissazione <ol><li> Stendere il midollo spinale isolato in un nuovo piatto di Petri rivestito con silicone contenente PBS, con perni di tenerlo, dal romboencefalo alla coda produrre un piano di montaggio preparazione. </li><li> Versare il PBS dal piatto e sostituirlo con 4% paraformaldeide in PBS. </li><li> Incubare a temperatura ambiente per 1 ora. </li></ol> Immunoistochimica <ol><li> Trasferire il midollo spinale fisso in una fiala contenente l&#39;anticorpo primario in PBS. </li><li> Incubare con agitazioni delicato per tutta la notte a 4 ° C. </li><li> Lavare l&#39;anticorpo X5 volte con PBS, ogni lavaggio per 1 ora con agitazioni gentile. </li><li> Aggiungi anticorpo secondario e incubare con agitazioni delicato per tutta la notte a 4 ° C. </li><li> Lavare l&#39;anticorpo X5 volte con PBS, ognilavaggio per 1 ora con agitazioni gentile. </li></ol> Montaggio libro aperto per l&#39;imaging <ol><li> Macchia di grasso o silicone a forma di rettangolo su un vetrino e flebo PBS all&#39;interno. </li><li> Posizionare il midollo spinale allungato al centro del rettangolo utilizzando una pinzetta e tirare fuori il liquido attorno ad esso con una pipetta Pasteur. </li><li> Luogo 1-2 gocce di mezzo di montaggio sul midollo spinale e coprire con coprioggetto. Squash per evitare bolle d&#39;aria. </li><li> Tenere le diapositive buio a 4 ° C. </li><li> Ispezionare il midollo spinale usando la microscopia confocale. </li></ol>

<ol>
	<li>Zisman, S., 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=Proteolysis+and+membrane+capture+of+F-spondin+generates+combinatorial+guidance+cues+from+a+single+molecule.">Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule.</a> J Cell Biol. 178 (7), 1237-1249 (2007).</li><li>Arakawa, T., Iwashita, M., Matsuzaki, F., Suzuki, T., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paths,+elongation,+and+projections+of+ascending+chick+embryonic+spinal+commissural+neurons+after+crossing+the+floor+plate.">Paths, elongation, and projections of ascending chick embryonic spinal commissural neurons after crossing the floor plate.</a> Brain Res. 1223, 25-33 (2008).</li><li>Avraham, O., 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=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4 (1), 21 (2009).</li><li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers.">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29 (3), 123-132 (2001).</li><li>Reeber, S. L., 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=Manipulating+Robo+expression+in+vivo+perturbs+commissural+axon+pathfinding+in+the+chick+spinal+cord.">Manipulating Robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord.</a> J Neurosci. 28 (35), 8698-8708 (2008).</li></ol>

Elettroporazione di DNA plasmidico in embrione di pollo si evolve come una tecnica potente per in vivo espressione ectopica. La combinazione di stimolatori specifici, ed elettroporazione pulcino fornisce uno strumento rapido ed efficace per decifrare le vie assonale di un gruppo di neuroni geneticamente definito 1,3,5. Utilizzando la Cre / loxP ed i sistemi di amplificazione Gal4/UAS può aumentare i livelli e la durata di espressione. Il quadro che emerge è di una divergenza complesso di spunti assonale che...

deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation

Impiego di elementi enhancer di guidare l&#39;espressione di geni reporter nei neuroni è un paradigma ampiamente utilizzato per il monitoraggio proiezione assonale. Per il monitoraggio proiezione assonale degli interneuroni spinali nei vertebrati, della linea germinale mirati geni reporter indicazione del tenore simmetria bilaterale. Pertanto, è difficile distinguere tra il ipsi-e contro-lateralmente assoni proiezione. Elettroporazione unilaterale nel tubo neurale pulcino fornisce un mezzo utile per limitare l&#39;espressione di un gene reporter per una parte del sistema nervoso centrale, ed a seguire proiezione assonale su entrambi i lati<sup&gt; 1 ,2-5</sup&gt;. Questo video mostra come gestire prima le uova prima dell&#39;iniezione. In fase HH 18-20, il DNA è iniettato nel livello sacrale del tubo neurale, poi elettrodi di tungsteno sono posti in parallelo per l&#39;embrione e brevi impulsi elettrici vengono somministrati con un generatore di impulsi. L&#39;uovo è sigillato con nastro adesivo e rimesso in un incubatore per un ulteriore sviluppo. Tre giorni dopo (E6) il midollo spinale viene rimosso come un libro aperto preparazione dall&#39;embrione, fissi, e trattati per tutta la colorazione degli anticorpi mount. Il midollo spinale è macchiato montato sulla slitta ed è visualizzato mediante microscopia confocale.

Watch this Scientific Journal Video about Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation at JoVE.com

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Questo video viene illustrato come visualizzare i percorsi assonale di gruppi geneticamente definita di neuroni nel midollo spinale embrionale pulcino utilizzando<em&gt; In ovo</emElettroporazione&gt; di geni reporter sotto il controllo di specifici elementi enhancer.

Decifrare Percorsi assonale di gruppi geneticamente definito di neuroni nel tubo neurale Chick Utilizzando In ovo Elettroporazione

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

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Research

JoVE Journal

Biology

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

9.2K Views.  Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School. Questo video viene illustrato come visualizzare i percorsi assonale di gruppi geneticamente definita di neuroni nel midollo spinale embrionale pulcino utilizzando In ovo di geni reporter sotto il controllo di specifici elementi enhancer.

Video: Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

In Electroporations Ovo della Fase HH 10 embrioni di pollo

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Ricerca

Biologia

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Elettroporazione di micobatteri

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Test di induzione neurale nell&#39;embrione Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria e elettroporazione plasmidi dell&#39;Organo murini di Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Immagini dal vivo della motilità cellulare e citoscheletro dei singoli neuroni e cellule della cresta neurale in embrioni di zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Monitoraggio Deformazioni tessuto morfogenetica in embrione di pollo primi

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

In elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...