Este trabalho foi financiado por uma doação da Fundação Alemã de Pesquisa (DFG; LU1455/1-1).

1. Cultura Ex ovo <ol><li> Frescas ovos fertilizados são colocados em seu lado mais longo em uma forçada projecto incubadora (BSS160, Ehret, Alemanha) a 37,5 ° C com umidade de 60% para a incubação. Ovos armazenados a 12 ° C durante mais de uma semana não deve ser utilizado. </li><li> Após a incubação durante 2,5 dias (cerca de estágio HH17), retire os ovos e rotular o topo com um lápis para indicar a direção para quebrar. </li><li> Antes de craqueamento, verter cerca de 20 ml de água destilada estéril para uma placa de Petri limpo grande (diâmetro de 145 mm; Greiner Bio-One GmbH, Alemanha). </li><li> Coloque outra pequena placa de Petri estéril (diâmetro de 94 mm; Greiner Bio-One GmbH) para a placa de Petri grande. </li><li> Quebrar os ovos na parte inferior contra uma ponta de metal afiada, abra cuidadosamente os ovos e transferir todo o conteúdo de cada ovo em placa de Petri pequena. </li><li> Cubra a placa de Petri grande com tampa e colocá-lo em outra incubadora (BINDER GmbH, Tuttlinge, Alemanha)para a cultura ainda mais a 37,5 ° C com cerca de 60% de humidade. Após incubação durante o número desejado de dias, os embriões podem ser utilizados para electroporação. </li></ol> 2. Preparação para Eletroporação ovo ex <ol><li> Puxe capilares de vidro com um puxador de microeletrodos (PUL-100 Micropipeta Extrator; Instrumentos de Precisão do Mundo, Berlim, Alemanha), utilizando tubos de vidro de 1,0 mm de diâmetro (TW100F-4; Instrumentos de Precisão do Mundo) e quebrar a ponta dos capilares de vidro em um apropriado diâmetro. </li><li> Configurar parâmetros apropriados (ou seja, as tensões diferentes para os diferentes tecidos e tamanho de embriões) para electroporação e ligar eléctrodos para um electroporator (CUY21-Edit; Nepa Gene, Chiba, no Japão) de acordo com os tecidos alvo e tamanho de embriões. </li><li> Preparar uma solução de plasmídeo contendo plasmídeos em vector pCAGGS que codificam um gene alvo, por exemplo, cadherin7 (Cad7; pCAGGS-Cad7 com uma concentração de 2,0 ug / uL) e um gene marcador,por exemplo, proteína fluorescente verde (GFP; pCAGGS-GFP com uma concentração de 0,25 ug / uL). Em seguida, adicionar Verde Rápido com uma concentração final de 0,1% (Sigma) para etiquetar a solução de plasmídeo com a cor verde. </li><li> Carregar o capilar de vidro com a solução de plasmídeo utilizando uma pipeta de boca. </li></ol> 3. Transferência de genes em Tectum Frango Óptica por Eletroporação ovo ex <ol><li> Após a cultura in ovo ex, por exemplo, por dia de incubação (E) 6, os embriões de galinha são utilizados para electroporação in ovo ex. </li><li> Cuidadosamente rasgar as membranas vitelinas e âmnio sobre o tecto óptico com fórceps finos e adicionar 3 gotas de solução de cloreto de sódio a 0,9% estéril sobre a superfície do tecto. </li><li> Injectar a solução de plasmídeo para dentro da cavidade do tectum por capilar de vidro utilizando a pipeta boca. </li><li> Coloque os eletrodos com uma agulha de tungstênio catodo e um anodo placa retângulo (cuy 661-3x7, Gene Nepa) ao lado do tecto, eimediatamente aplicar impulsos eléctricos (seis impulsos, 25 V, 60 ms de comprimento de pulso, 100 intervalos de ms em cada caso) produzidas pelo electroporator ao tectum. </li><li> Cobrir a placa de Petri de grandes dimensões com a tampa e retorná-lo para a incubadora. Após incubação durante dias adequadas (por exemplo, 2 ou 3 dias após a electroporação), o tecto são recolhidos e fixado para imunocoloração (Figura 1) e detecção biológica. </li></ol> 4. Os resultados representativos Superexpressão bem sucedida das proteínas exógenas de Cad7 e GFP por electroporação in ovo ex é mostrado na Figura 1 como um exemplo. Quando Cad7 juntamente com o plasmídeo GFP plasmídeo foi electroporado para o tectum em E6, dois ou três dias após a electroporação os embriões foram recolhidas e fixada. Em E8, a proteína GFP é fortemente expresso na tectum em imagens inteiras de montagem (Figura 1A-C). Além disso, nas secções do tectum em E9,GFP proteína (verde na Figura 1D) e Cad7 proteína (vermelho na Figura 1E) são co-expressos (amarelo na Figura 1F), sugerindo que a electroporação in ovo ex é um método bem sucedido para a transferência de genes em que os embriões de galinha mais velhos in vivo. <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="A Figura 1"/> Figura 1. Quando a plasmídeos de codificação da GFP e Cad7 são cotransfectados para o tectum galinha no E6, dois ou três dias após a electroporação in ovo ex, proteína GFP (verde) é fortemente expresso como mostrado nas imagens wholemount (AC) em E8. Em secções do tectum em E9, GFP proteína (verde em D) e Cad7 proteína (vermelho em E) são co-expressos (amarelo em F). BF, imagem de campo claro. Barra de escala: 200 um em um para AC; 100 mm em D para DF.

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</li><li>Hamburger, V., Hamilton, H. 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> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

Não há conflitos de interesse declarados.

Este protocolo fornece um guia para a transferência de genes em embriões de galinha mais velhos (por exemplo, para o tecto óptico em E6) por electroporação in ovo ex. Este método estende a utilização de electroporação de embriões de galinha mais velhos e pode ser facilmente aplicado a muitos laboratórios para estudar a função do gene in vivo em fases posteriores. Embriões de E4, a pelo menos E7 pode com sucesso ser electroporado por este método e os embriões eletroporados pode ser sobr...

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Electroporator </td><td> Nepa Gene, Japão </td><td> CUY21-Edit </td></tr><tr><td> Eletrodos </td><td> Nepa Gene, Japão </td><td> CUY661-3x7 </td></tr><tr><td> Ovo incubadora </td><td> Ehret, Alemanha </td><td> BSS160 </td></tr><tr><td> Embrião incubadora </td><td> BINDER GmbH, Alemanha </td><td></td></tr><tr><td> Microscópio fluorescente </td><td> Keyence, Deutschland GmbH </td><td> BZ-8000 </td></tr><tr><td> Placa de Petri </td><td> Greiner Bio-One GmbH, Alemanha </td><td></td></tr><tr><td> Extrator de microeletrodos </td><td> Mundo Precision Instruments, Alemanha </td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

O embrião de galinha fornece um sistema modelo excelente para estudar a função e regulação de genes durante o desenvolvimento embrionário. Em electroporação in ovo é um método poderoso para sobre-expressar genes exógenos ou para baixo-regular os genes endógenos in vivo em embriões de galinha 1. Diferentes estruturas, tais como genes de DNA plasmídeos codificando 2-4, pequena interferência RNA (siRNA) plasmídeos 5, pequeno sintético RNA oligos 6, e os oligonucleótidos anti-sentido morfolino 7 pode ser facilmente transfectado em embriões de galinha por electroporação. No entanto, a aplicação de eletroporação em ovo é limitada aos embriões em estágios iniciais de incubação (menores de estágio HH20 - de acordo com Hamburgo e Hamilton) 8 e existem algumas desvantagens para a sua aplicação em embriões em estágios mais avançados (com mais de estágio HH22 - cerca de 3,5 dias de desenvolvimento). Por exemplo, a membrana vitelina em fases posteriores é usualiado preso à membrana devem e abrir uma janela no shell provoca ruptura dos vasos, resultando na morte dos embriões; embriões mais velhos são cobertos por vitelina e alantóide navios, onde é difícil de acessar e manipular os embriões; embriões mais velhos mover vigorosamente e é difícil de controlar a orientação através de uma janela relativamente pequena na concha. Neste protocolo, demonstramos um método de eletroporação ex ovo de transferência de genes em embriões de galinha em estágios mais avançados (com mais de estágio HH22). Para eletroporação in ovo ex, os embriões são cultivadas em placas de Petri de 9 e vitelina e alantóide embarcações são amplamente difundidos. Sob estas condições, os embriões de galinha mais velhos são facilmente acessíveis e manipulado. Portanto, este método supera as desvantagens de ovo em electroporação aplicado aos embriões de galinha mais velhos. Usando este método, os plasmídeos podem ser facilmente transfectado em diferentes partesdos embriões de galinha mais velhos 10-12.

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Um método de transferência de genes em embriões de galinha em fases posteriores de incubação (com mais de Hamburger e Hamilton fase (HH) 22) é descrito. Este método supera as desvantagens dos<em&gt; In ovo</emEletroporação&gt; aplicado aos embriões de galinha e mais velhos é uma técnica útil para estudar a função e regulação gênica em estádios mais avançados de desenvolvimento.

Transferência de Embriões em Gene de frango por mais velhos<em&gt; Ovo ex</em&gt; Eletroporação

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K visualizações.  School of Medicine University of Rostock. Um método de transferência de genes em embriões de galinha em fases posteriores de incubação (com mais de Hamburger e Hamilton fase (HH) 22) é descrito. Este método supera as desvantagens dos In ovo aplicado aos embriões de galinha e mais velhos é uma técnica útil para estudar a função e regulação gênica em estádios mais avançados de desenvolvimento.

Vídeo: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Transferência nuclear em oócitos de camundongos

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Biologia

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero injeção intraventricular e eletroporação de embriões de ratos E16

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero injeção intraventricular e eletroporação de embriões de camundongos E15

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Fator de Crescimento colocando-Revestido Beads em embriões de galinha Early Stage

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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.

Em Electroporations Ovo de Estágio HH 10 embriões de galinha

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

No útero e ex eletroporação in vivo para Expressão Gênica em células ganglionares da retina do rato

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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.

Pintinho Ex ovo Cultura e Ex ovo CAM Ensaio: como ele realmente funciona

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...