Name: in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations
Uploaded: 2012-10-16T12:00:00
Description: Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

Работа в лаборатории ES при поддержке Швейцарского национального фонда науки. Мы хотели бы поблагодарить д-ра Удар Kunz за помощь в съемках.

1. Подготовка RNAi плазмидной ДНК для сотовых Тип конкретных генов Плазмиды (рис. 1) синтезируется с использованием стандартных методов молекулярной клонирование, как ранее подробно описана 3,4. 1,1 клонирования в векторах: oligonucelotide дизайн <ol><li> М...

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	<li>Ch&#233;dotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Current Opinion in Neurobiology. 21, 68-75 (2011).</li><li>Avraham, O., Hadas, Y., Vald, L., Zisman, S., Schejter, A., Visel, A., Klar, A. <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 Development. 4, 21 (2009).</li><li>Wilson, N. H., Stoeckli, E. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-92 (1951).</li><li>Boulland, J., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+Human+Stem+Cells+into+the+Chicken+Embryo.">Xenotransplantation of Human Stem Cells into the Chicken Embryo.</a> J. Vis. Exp. (41), e2071 (2010).</li><li>Kim, B. G., Dai, H. -. N., McAtee, M., Vicini, S., Bregman, B. 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L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Li, X., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase+expression+in+the+floorplate,+notochord+and+gut+epithelium+in+transgenic+embryos+driven+by+the+Hoxa-1+enhancer+III.">Cre recombinase expression in the floorplate, notochord and gut epithelium in transgenic embryos driven by the Hoxa-1 enhancer III.</a> Genesis. 26, 121-122 (2000).</li><li>Mauti, O., Baeriswyl, T., Stoeckli, E. T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+by+Injection+and+Electroporation+of+dsRNA+in+Avian+Embryos.">Silencing by Injection and Electroporation of dsRNA in Avian Embryos.</a> Cold Spring Harbor Protocols. , (2008).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Developmental Dynamics. 229, 433-439 (2004).</li><li>Cullen, B. 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Этот простой, векторную искусственных стратегия выражение микроРНК могут быть использованы для нокдаун эндогенной экспрессии генов в куриных нервной трубки. Эти функциональные инструменты предлагают несколько глушителей гена, временной контроль и камерного типа специфичности, что?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td></tr><tr><td> 0,5 мм стеклянных капилляров </td><td> Инструменты Всемирной Precision </td><td> 1B120F-4 </td></tr><tr><td> Стеклянные иглу съемника </td><td> Narishige </td><td> PC-10 </td></tr><tr><td> Электропоратора </td><td> BTX </td><td> ECM 830 </td></tr><tr><td> Sylgard силиконового эластомера </td><td> Инструменты Всемирной Precision </td><td> SYLG184 </td></tr><tr><td> Вольфрамовой проволоки, 0,075 мм </td><td> Инструменты Всемирной Precision </td><td> TGW0325 </td></tr><tr><td> Насекомое булавки, 0,20 мм </td><td> Средства изобразительных наук </td><td> 26002-20 </td></tr><tr><td> Насекомое булавки, 0,10 мм </td><td> Средства изобразительных наук </td><td> 26002-10 </td></tr><tr><td> Весна ножницы </td><td> Изобразительное наукиолы </td><td> 15003-08 </td></tr><tr><td> Дюмон # 5 щипцы </td><td> Средства изобразительных наук </td><td> 11252-20 </td></tr><tr><td> Дюмон № 55 щипцы </td><td> Средства изобразительных наук </td><td> 11255-20 </td></tr><tr><td> Быстрый DiI </td><td> Молекулярные зонды </td><td> D-7756 </td></tr><tr><td> Флуоресцентные микроскопы </td><td> Олимп </td><td> SZX12, BX51 </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

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

Метод, которым экспрессию генов в нервной трубке может быть подавляется в клетку определенного типа, прослеживается образом описано. Мы показываем, как<em&gt; В ово</em&gt; Электропорации микроРНК основе плазмид, которые вызывают spatiotemporally контролируемых РНК-интерференции может быть использован для исследования спаечный руководством аксонов в развивающейся нервной трубки.

В ово Electroporation микроРНК основе плазмид в развивающихся нервной трубки и оценки фенотипов по DiI инъекций в открытом книгу Препараты

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

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Neuroscience

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

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