Este trabalho foi apoiado pelo Deutsche Forschungsgemeinschaft (DFG), queremos agradecer Ayele Tsedeke Taddese para suporte técnico.

1. Preparação das agulhas de vidro (Figura 1) <ol><li> Use capilares de vidro com um diâmetro 0,75 milímetros para preparar as agulhas de injeção (Figura 1A). </li><li> Colocar o capilar de vidro, em um extrator de agulha e puxe a agulha com o seguinte parâmetro: valor ciclo de aquecimento: 463; puxando valor ciclo: 230; velocidade: 150 ms; tempo: 150 mseg (Figura 1B). </li><li> Quebre o capilar de vidro puxado com uma pinça relojoeiro para produzir uma agulha de 20 um de diâmetro sob um estereoscópio com uma ocular micrométrica. </li><li> Use um torno com uma roda de fiar de borracha molhada para aguçar a agulha e produzir a 20 mM bisel (Figuras 1C e 1D). Nota: Sharp, agulhas biseladas melhorar a facilidade de inserção no interior do ventrículo e, portanto, minimizar o dano ao tecido e permite que o músculo perfurada para selar após a remoção da agulha (figuras 1E-G). </li></ol> 2. Preparção da Solução Morfolino <ol><li> Prepare o estoque morfolino dissolvendo o morfolino liofilizado em 1x com fosfato tamponado salino (PBS) a uma concentração final de 7,5 mM. [Veja as instruções do fabricante para obter mais detalhes (tabela Materiais)]. </li><li> Preparar a solução de injeção morfolino misturando solução estoque morfolino 2,5 mL (7,5 mM) com 2,8 mL solução Endo-Porter estoque (1 mM) (Ver tabela de materiais) para uma concentração final de 3,5 mM morfolino e 0,5 mM Endo-Porter. </li></ol> 3. Preparação de Injecção Morfolino (Figura 2) <ol><li> Coloque a agulha de vidro chanfrado com 5 mL desta solução, utilizando uma micropipeta com a 10 mL microloader ponteira. </li><li> Inserir a agulha de vidro no suporte de agulha do micromanipulador ligado à bomba de pico pneumático (Figuras 2A-C). </li><li> Coloque o suporte da agulha ao lado do microscópio de modo que a agulha apenas as necessidadespara ser movido numa direcção planar a inseri-lo no interior do ventrículo cardíaco da larva (Figura 2A). </li><li> Ajuste o ângulo para as injeções em torno de 45 °. </li><li> Defina os valores microinjetor como segue: pressão de recalque: 20 libras por polegada quadrada (psi); pressão de ejeção: 15 psi; 100 ms gama de gating, valor do período de 1,9 (corresponde a 10,9 ms). </li><li> Melt agarose em 1x PBS utilizando um forno de microondas padrão para produzir 20 ml de 1,5% (w / v) de gel. Despeje gel derretido em um prato de 10 centímetros de Petri. Coloque uma forma de injeção ranhuras no agarose quente, de modo que uma vez endurecido, o agarose terá sulcos em que a larva sedado serão colocados 11 (Figuras 2D e 2E). </li></ol> 4. Injecções (Figura 3) <ol><li> Anestesiar larva em 100 ml de água do aquário com 20 mg / L tricaina (sulfonato L-etil-m-amino-benzoato de-metano) até que pare de responder ao toque. </li><li> Use uma pipeta Pasteur de plásticoe transferir cuidadosamente a larva sedado dentro de uma ranhura do molde de agarose molhado de modo que o lado ventral está virada contra a parede vertical do sulco de agarose (Figura 3A). </li><li> Colocar a placa de agarose sob o microscópio estereoscópico, de modo que o ventrículo está voltado para fora a partir da agulha de injecção (Figura 3A). </li><li> Abaixe a agulha para inseri-lo para o ventrículo cardíaco. Insira a agulha apenas 1-2 mM para o ventrículo tomando cuidado para não inserir a agulha muito profundamente (Figura 3B e 3C). </li><li> Uma vez que a agulha é inserida, injectar a solução de morfolino no ventrículo com 4-6 pulsos, cada um fornecendo 3 nl da solução, com intervalos de espera para permitir a compensação do coração (Figuras 3D e 3E). </li><li> Após a injecção, retirar a agulha (paralelo ao plano de inserção) e, em seguida, transferir cuidadosamente a larva usando uma pipeta de Pasteur de plástico cheio com meio E3 back em uma placa de Petri contendo meio fresco E3. </li><li> Coloque a agulha em uma placa de Petri contendo 1x PBS para impedir a secagem da agulha durante a transferência da larva injectado. </li><li> Repita os passos 4,1-4,6 cada 12-24 horas, para a duração da experiência. Nota: Repetindo as injecções assegura a captação e manutenção da morfolino em células. </li></ol> 5. Análises de injecções (Figura 4) <ol><li> Anestesiar os peixes em 100 ml de água do aquário com 20 mg / L tricaina até que parar de responder ao toque. </li><li> Coloque a larva lateralmente em uma superfície plana molhada 1,5% (w / v) placa de agarose cobertas com meio E3. </li><li> Larvas de imagem usando um microscópio estereoscópico com claro e imagens de fluorescência. Observação: Como as larvas são transparentes, o morfolino marcado com fluoresceína deve ser visível como verde fluorescente nos vasos sanguíneos em todo o animal imediatamente após a injecção. Esta fluorescência irá dispersar ainda mais nos tecidos vascularizados pela15 minutos (Figuras 4A-4C). </li></ol> 6. Avaliação da Regenerativa Conseqüência <ol><li> Avaliar as imagens capturadas usando Fiji Imagem J software livre (http://fiji.sc/Fiji). Para investigar a regeneração, use a ferramenta de rastreamento de linha para determinar a quantidade de crescimento regenerativo que ocorreu em controle e injeção de larva morfolino. </li><li> Para calibrar imagens, abrir um dos arquivos de imagem originais em Fiji por arrastar e soltar o arquivo para a janela principal de Fiji. </li><li> Para definir a escala para todas as imagens a seguir, vá para &quot;Analisar&quot; no menu principal. </li><li> No menu drop-down, clique em &quot;Definir escala&quot;. </li><li> Nesta janela, ative a opção &quot;Global&quot;, clicando na caixa de seleção. </li><li> Agora, abra a imagem para medir a quantidade de crescimento regenerativo novamente por arrastar e soltar (veja o passo 6.1). </li><li> Escolha a ferramenta &quot;seleções à mão livre&quot; na barra de ferramentas. </li><li> Cercar a área a ser medida (Figuras 4J-4D). </li><li> Pressione Ctrl + M. Isto irá abrir uma nova janela &quot;Results&quot;, mostrando o valor da área cercada em pixels quadrados. </li></ol>

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A injeção intraventricular fornece um método de avaliação rápida e confiável para testar a função do gene em fases posteriores do desenvolvimento ou da homeostase corporal sem afetar a função dos genes durante a embriogênese. Para garantir o sucesso desta técnica, deve-se estar ciente de quatro pontos fundamentais: 1) tamanho da agulha, 2) secagem para fora da agulha, 3) minimizar o volume, e 4) tempo de exposição mínimo na solução de sedação. Agulhas que são demasiado pequenos vai entupir com freq?...

A regeneração de órgãos e apêndices é de fundamental importância para a sobrevivência e fitness; no entanto, vários vertebrados, incluindo o homem limitaram capacidades regenerativas. Embora existam vários modelos animais que têm ampla capacidade regenerativa, inverter técnicas genéticas para avaliar a função do gene durante o órgão e regeneração apêndice continuam a ser muito limitada ou inexistente. Por conseguinte, novas abordagens são necessárias para dissecar a biologia molecular de regeneração nestes organismos modelo. O peixe-zebra é um modelo bem estabelecido para a compreensão da biologia celular e molecular do órgão e regeneração apêndice 1, não só devido à sua significativa capacidade de regenerar vários órgãos, tecidos e apêndices, mas também porque existem várias linhas de peixes transgênicos para rastrear células e para sobre-expressar genes constrói 2, 3. No entanto, a inibição do gene em peixes-zebra larval é limitada principalmente ao overexpression de construções dominantes-negativas, que não estão disponíveis para todos os genes de interesse ou cujo produto transgene pode adquirir efeitos ganho de função que não refletem a atividade endógena do gene. Assim, um método alternativo para remover especificamente expressão gênica por nocaute ou nocaute é necessária para superar esses problemas. Específica do gene alvo usando TALENS existe como um meio genéticos reversa para nocautear a função do gene; no entanto, esta estratégia de nocaute é muito frequentemente limitado a avaliação funcional durante a embriogênese cedo, porque os requisitos iniciais do gene impedir a progressão do desenvolvimento embrionário. Assim, o estudo de fenômenos posteriores, como a regeneração ou a homeostase do órgão após o desenvolvimento usando TALENS é impedida 4, 5. Portanto, uma estratégia alternativa de remoção de gene é necessário que atinge a função do gene após o desenvolvimento cedo para avaliar os requisitos de genes em órgãos e estruturas totalmente formados. </p&gt; Morfolino injecção foi demonstrado ser eficaz no direccionamento de genes em alguns órgãos adultos e adultos regenerar barbatana 6-8, mas estes métodos requerem a electroporação e muitos órgãos internos são difíceis para electroporar, quer devido à sua localização, ou devido à sua sensibilidade à eléctrica perturbação. Além disso, alguns tecidos, em que a larva são difíceis de administrar directamente, devido à injecção directa pode perturbar a sua integridade estrutural ou porque o seu tamanho é limitante. O caudal da larva é uma estrutura deste tipo, porque a injecção directa na membrana embrionária não é possível. Assim, foi necessária uma alternativa para eletroporação e injeção direta para atingir genes em tecidos que são demasiado pequenos para injetar ou não pode ser eletroporados. A fim de orientar e inibir a função de genes específicos durante a regeneração da nadadeira caudal larval, modificamos tecnologias morpholino existentes permitindo a umAVALIAÇÃO da função do gene durante a regeneração da nadadeira caudal em larva late-encenado. Este método emprega entrega intraventricular 9 de fluoresceína-marcado morpholinos juntamente com Endo-Porter reagente de transfecção 10. Uma vez no interior do ventrículo, a mistura de morfolino-Endo-Porter espalha-se rapidamente por toda a larva através da vasculatura e entra tecidos que tenham sido previamente impossível de atingir. Este método de injecção pode ser modificado para alvejar genes em tecidos específicos e, possivelmente, pode ser aplicado em outros modelos animais, que não têm ainda métodos de genética inversa para inibir a função do gene. Assim, oferece um método rápido e fácil com o potencial para uso amplo leque de estudar imediatamente a função do gene durante a homeostase órgão geral e regeneração em estágios larvais.

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		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
	</tbody>
</table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

O peixe-zebra é um modelo importante para entender a biologia celular e molecular do órgão e regeneração apêndice. Contudo, as estratégias moleculares para empregar genética reversa ainda não foram adequadamente desenvolvidos para avaliar a função do gene em regeneração ou homeostase do tecido durante a fase larval após embriogênese peixe-zebra, e vários tecidos dentro do larva zebrafish são difíceis de atingir. Injecções intraventriculares morpholinos gene-específicos oferecer um método alternativo para a actual incapacidade para alvejar genes genomicamente de peixes-zebra de uma forma temporal controlada nestas fases. Este método permite a completa dispersão e subsequente incorporação da morfolino em diversos tecidos em todo o corpo, incluindo as estruturas que eram anteriormente impossíveis de alcançar, tais como os do caudal das larvas, de uma estrutura, muitas vezes utilizados para pesquisar de forma não invasiva a regeneração do tecido. Vários genes ativados durante a regeneração membrana embrionária larval também são apresent na regeneração de tecidos adultos de vertebrados, de modo a larva é um modelo útil para compreender a regeneração em adultos. Este método de dispersão morfolino permite a identificação rápida e fácil dos genes necessários para a regeneração dos tecidos das larvas, bem como outros fenómenos fisiológicos que regulam a homeostase do tecido após a embriogénese. Portanto, este método de entrega fornece uma estratégia atualmente necessário para controle temporal para a avaliação da função do gene após a embriogênese.

O forte, agulha de injeção chanfrado é facilmente colocado no ventrículo cardíaco da larva zebrafish quando foi abordado dorsal (Figura 3A). O coração continua a bombear e o fluxo de sangue é mantido apesar da presença da agulha (figuras 3B e 3C). Injeções cuidadosas não perturbar a morfologia do ventrículo ou contrações cardíacas (Figura 3D), apesar de a injeção do morfolino para o coração (Figura 3E). <p class=...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Um método de genética reversa adaptável para peixe-zebra para avaliar a função do gene em fases posteriores do desenvolvimento e homeostase fisiológica, como a regeneração do tecido usando injeções intraventricular de morpholinos gene-específicos.

Reverter Genética Abordagem Morfolino Usando Injeção Cardíaca Ventricular para transf múltiplos tecidos de difícil alvo do Zebrafish Zergling

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K visualizações.  Technische Universität Dresden.  Um método de genética reversa adaptável para peixe-zebra para avaliar a função do gene em fases posteriores do desenvolvimento e homeostase fisiológica, como a regeneração do tecido usando injeções intraventricular de morpholinos gene-específicos. 

Vídeo: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microinjeção de mRNA e Oligonucleotídeos Anti-Senso Morpholino em embriões Zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biologia

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Injection Ventrículo Cerebral

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbital de injeção no Zebrafish Adulto

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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

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

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Uma Abordagem Prática para mapeamento genético induzível Fate: Um Guia Visual para Mark e Track Células In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

A abordagem inversa Genética para Teste de Redundância funcional durante a embriogênese

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injeção intraperitoneal em Zebrafish Adulto

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish Adulto

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Um multi-furo criotubo Elimina congelação Artifacts quando tecidos musculares são directamente Imerso em azoto líquido

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...