Bu çalışma, İsrail Bilim Vakfı, İsrail sağlık bakanlığı, ve DFG (Alman Araştırma Vakfı) AK hibe tarafından desteklenen oldu.

I. Elektroporasyon Yumurta taşıma <ol><li> Nemlendirilmiş bir inkübatör seti 37-38 ° C, tercih sallanan tepsileri bir kuluçka yumurtaları yerleştirin. </li><li> Hamburger ve Hamilton (HH) evre 18-20 ulaştığınızda Embriyo, kuluçka yaklaşık 66 saat sonra electroporated. Bu aşamada, baş, gövde (servikal eğme olarak adlandırılan durum) ve ön, arka, sağ ve sol vitellin venlerin görülmesi gereken ekstra embriyonik kan damarları, geniş bir dizi doğru açılarda yatıyor. Bu arttırıcı bağımlı spesifik ifade 1,3,4 için optimal bir aşamasıdır. Uygulanabilir ve senkronize embriyolar elde etmek için, inkübatör sıcaklık ve nem izlemek için önemlidir. </li><li> Inkübasyon 66 saat sonra, yumurtalar inkübatör çıkarın. Yumurta yatay olarak yerleştirin. 5-10 dakika bekleyin. Embriyo artık yumurta üst kutbunda yer almaktadır. </li></ol> Hazırlıklar <ol><li> Hank çözümü penisilin / streptomisin (P / S) (1:100 dilüsyon) ile hazırlayın. </li><li> Microcapillary cam kılcal damarlar (0.5 mm çap) çekin. </li><li> Mikromanipülatör içine elektrotlar (tungsten) yerleştirin ve darbe jeneratörü (ECM 830) elektrotlar bağlamak. </li><li> - 3, darbe uzunluğu 50 ms voltaj 30v, bakliyat sayısı: Aşağıdaki parametreler ile darbe jeneratörü ayarlayın. </li><li> Ağız pipet, bir şırınga iğnesi ve bir kaset hazırlayın. </li><li> DNA (2-5μg/μl) istenen bir karışım hazırlayın ve Hızlı Yeşil boya ekleyin. </li></ol> Pencereleme ve elektroporasyon <ol><li> Bir şırınga kullanarak, her bir yumurta 5-6 ml albümin çıkarın. </li><li> Yumurtanın üst tarafında küçük bir makas bir oval pencere açıkken. </li><li> 0.5-1ml Hank + P / S çözümü ile Nemli embriyo. </li><li> Cam DNA karışımı ile microcapillary yüklemek için, bir ağız pipet kullanın. </li><li> Size doğru kuyruk ile embriyo yerleştirin. Bu aşamada, embriyo omurilik açıkça görülür. Bu nedenle, hiçbir karşıt teknikler gereklidir. Omurilik iki ucunda, baş ve kuyruk mühürlenmiş, ancak sizin microcapillary yeterince ince ise, küçük bir delik ponksiyon ve sığ bir açıyla nöral tüp nüfuz edecektir. Sağ çapı microcapillary DNA ile zarar vermeden tüpün içine nüfuz ve düzenli bir soluk verme ile DNA serbest kolayca yüklenmiş olması gerekir. </li><li> Ağız pipet kullanarak DNA enjekte edilir. Yeşil boya, gelişmekte olan beynin veziküller kuyruk ucu yaymak gerekir. </li><li> Tüp kendisi ve nabız dokunmadan mümkün olduğunca yakın, nöral tüp paralel elektrotlar yerleştirin. Darbe zamanda, elektrotlar elektriksel iletkenlik sağlayan sıvı ile kaplı olmalıdır. Genellikle, Hank yalvarıyor eklendi miktarı yeterlidir. Eğer değilse bir damla Hank darbe hemen önce ekleyin. </li><li> Elektrotlar dikkatlice çıkarın ve bir bant ile pencere mühür. Yumurta tamamen sızdırmazlık için aşağıdaki kuluçka dönemi sırasında embriyo kurumasını önlemek için önemlidir. </li><li> Inkübatör yumurta geri yerleştirin. </li></ol> II. Omurilik açık-kitap hazırlanması Embriyo omurilik Dekolmanı <ol><li> Yumurta, E6, eletroporated embriyo çıkarın ve PBS içeren silikon kaplı bir Petri kabı yerleştirin. </li><li> Membranlar kesip ve ventral tarafta tutun pimleri kullanarak embriyo streç. </li><li> Keskin bir tungsten kullanma Beyin kuyruk aşağı, çatı levha boyunca uzunlamasına bir kesi yapmak microcapillary. </li><li> Omurilik dorsal kök gangliyon (DRG) ayrılmakta, omuriliğin her iki tarafında ek bir iki uzunlamasına insizyonlar olun. </li><li> Omurilik zedelemeden, kuyruk Beyin başlayan doku taban plakası ile ayırın. </li><li> Ince makas kullanarak Beyin omurilik enine bir bölümünde kesin ve vücuttan ayrı. </li></ol> Tespit <ol><li> Silikon düz bir montaj hazırlık üreten kuyruk Beyin tutun pimleri kullanarak, PBS içeren ile kaplı yeni bir petri izole spinal kord sürün. </li><li> Çanak PBS dökün ve fosfat tamponlu salin içinde% 4 paraformaldehid ile değiştirin. </li><li> 1 saat oda sıcaklığında inkübe edin. </li></ol> İmmünohistokimya <ol><li> Sabit omurilik PBS içinde primer antikor içeren bir şişeye aktarın. </li><li> 4 gece boyunca hafif ajitasyon ile inkübe ° C. </li><li> PBS ile antikor X5, süreleri, her yıkama nazik ajitasyon ile 1 saat yıkayın. </li><li> Sekonder antikor ekleyin ve 4 gece boyunca hafif ajitasyon ile inkübe ° C </li><li> Her antikor X5 kez PBS ile yıkayınnazik ajitasyon ile 1 saat süreyle yıkayın. </li></ol> Görüntüleme için açık bir kitap Montaj <ol><li> Smear gres veya silikon bir slayt üzerinde bir dikdörtgen şeklinde ve içinde PBS damla. </li><li> Cımbız ve Pasteur pipeti ile çevresinde sıvı çizmek kullanarak dikdörtgenin ortasında gergin omurilik yerleştirin. </li><li> Omurilik 1-2 damla montaj medya yerleştirin ve kapağı kayma ile kaplayın. Squash hava kabarcıklarını önlemek için. </li><li> 4 ° C&#39;de karanlıkta slaytlar tutun </li><li> Konfokal mikroskopi kullanılarak omurilik inceleyin. </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>

Civciv embriyo içine plazmid DNA Elektroporasyon in vivo ektopik ifade için güçlü bir teknik olarak gelişiyor. Özel arttırıcılar ve civciv elektroporasyon kombinasyonu nöronlar 1,3,5 genetik olarak tanımlanan bir grup aksonal yollar deşifre için hızlı ve etkili bir araç sağlar . Cre / LoxP ve Gal4/UAS amplifikasyon sistemlerinin kullanılması ifade düzeyleri ve süresini arttırır. Ortaya çıkan resim interneuron alt popülasyonlar, kendi aksonal projeksiyonlar yönü ile tanımlanan kay...

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

Nöronlarda muhabiri genlerin ifade sürücü İstihdam artırıcı unsurları aksonal projeksiyon izlemek için yaygın olarak kullanılan bir paradigma. Omurgalılarda spinal internöronlar aksonal projeksiyon izleme, germ line-hedefli muhabiri genler bilateral simetrik etiketleme verim. Bu nedenle, IPSI ve kontra-yanal projelendirme aksonlar ayırt etmek zordur. Civciv nöral tüp tek taraflı elektroporasyon, merkezi sinir sisteminin bir tarafı bir muhabir gen ifadesini sınırlamak ve her iki tarafta aksonal projeksiyon takip etmek faydalı bir araç sağlar<sup&gt; 1 ,2-5</sup&gt;. Bu video ilk enjeksiyon öncesinde yumurta nasıl işleneceğini gösteriyor. HH evre 18-20, DNA sakral nöral tüp içine enjekte edilir, daha sonra tungsten elektrotlar embriyo paralel olarak yerleştirilir ve kısa bir darbe jeneratörü elektrik darbeleri ile yönetilmektedir. Yumurta bandı ile kapatılmış ve daha da geliştirilmesi için bir inkübatör içine geri yerleştirilir. Üç gün sonra (E6) omurilik, embriyo açık bir kitap hazırlık olarak kaldırılır sabit ve tüm montaj antikor boyama için işlenmiş. Lekeli omurilik slayt monte edilir ve konfokal mikroskopi kullanılarak görüntülendi.

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

Bu video nöronların genetik olarak tanımlanan gruplar aksonal yollar kullanarak embriyonik civciv omurilik görselleştirmek için nasıl gösterir<em&gt; Ovo</em&gt; Muhabiri genlerin kontrolü altında belirli artırıcı unsurların elektroporasyon.

Nöronlar, Genetik Tanımlı Gruplar Aksonal tanımında kullanılması Civciv Nöral Tüp Deşifre Ovo Elektroporasyon

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 Görüntüleme.  Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School. Bu video nöronların genetik olarak tanımlanan gruplar aksonal yollar kullanarak embriyonik civciv omurilik görselleştirmek için nasıl gösterir Ovo Muhabiri genlerin kontrolü altında belirli artırıcı unsurların elektroporasyon.

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.

HH Sahne 10 Tavuk Embriyolar Ovo Electroporations

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 ...

Biyoloji

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.

Mikobakteriler, Elektroporasyon

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.

Civciv Embriyo Sinir İndüksiyon için Testi

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.

Civciv Ex ovo Kültür ve Ex ovo CAM Testi: Gerçekten Nasıl Çalışır?

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.

Elektron Mikroskobu için embriyo Hazırlık Drosophila Embriyonik kalp tüpü

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.

İlköğretim Kültür ve Corti murin Organ Plazmid Elektroporasyon.

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.

Zebra balığı Embriyolar Bireysel Nöronlar ve Sinir Crest Hücreler Aktin Sitoiskelet Hücre Motilite ve Canlı Görüntüleme

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.

Saflaştırılması M. magneticum Deformasyon AMB-1 Magnetosome İlişkili Protein 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.

Erken Civciv Embriyo morfogenetik Doku deformasyonlar Takibi

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.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı Retina

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