This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

Experiências com animais foram aprovados pelo comitê local do cantão de Berna Kantonstierarzt, Kantonaler Veterinärdienst Bern (BE85 / 15). 1. Preparação de Xenopus Os oócitos <ol><li> Manter rãs (Xenopus laevis) em uma de 12 h / 12 h de luz / escuro no ciclo de água, que é estritamente mantida a 20 ° C. </li><li> Retire os lobos dos ovários de rãs do sexo feminino 9. NOTA: A remoção dos lóbulos é um estímulo para a regeneração, e muitas vezes a qualidade dos oócitos melhora com a cirurgia. O oócito é coberto por uma camada vitelino, uma camada de células do folículo, e tecido conjuntivo contendo os vasos sanguíneos 21. Toda a estrutura é denominado o &quot;folículo.&quot; </li><li> Coloque os lobos dos ovários, que são densamente embalado com os folículos contendo os oócitos em estéreis de Barth Saline 7 (MBS) suplementado com penicilina e estreptomicina (NaCl 88 mM, KCl 1 mM, 2,4 mMNaHCO3, MgSO4 0,82 mM, CaCl 0,41 mM de 2, Ca 0,33 mM (NO 3) 2, 10 mM de ácido 4- (2-hidroxietil) -1-piperazina; HEPES (pH 7,5, NaOH), 100 ug / mL de penicilina, e 100 ug / mL de estreptomicina). </li><li> Single-out fase V-VI 21 folículos. Segurar o lóbulo ovário com uma pinça. Diminuir a ansa de platina (Figura 1) ao longo dos folículos e suavemente retirar o circuito para perturbar o tecido conjuntivo contendo o folículo do tecido do ovário. NOTA: Fase V-VI 21 folículos são caracterizados por seu tamanho (1,0-1,2 mm de diâmetro), e pelo bom contraste entre o hemisfério animais pigmentada escura e do hemisfério vegetal amarelada. </li><li> Transferir apenas folículos saudáveis de aspecto (isto é perfeitamente esféricas e com pigmentação intacta) para uma placa de Petri de diâmetro de 35 milímetros por meio de uma pipeta de Pasteur de plástico (cortar a parte de trás até um diâmetro de 1,5 milímetros de abertura). </li></ol> Nota: O sistema de microinjeção aqui descrito é derivado do que a relatada por Kressmann e Birnstiel 22. <ol><li> Prepare ARNc a partir dos respectivos ADNc que codificam para as subunidades da α 4 β 2 δ receptor GABA A por transcrição in vitro, a adição de um poli (A +) de cauda, e quantificação de ARN por electroforese em gel de 23. </li><li> Prepare pipetas de microinjecção de capilares de vidro de borossilicato (1,0 mm de diâmetro externo (OD), diâmetro interno de 0,58 mm (ID), 100 mm de comprimento) utilizando um extractor de micropipeta. Romper as pontas dos capilares de vidro sob um microscópio usando um micromanipulador e microfilamentos para criar um diâmetro da ponta de 12 - 15 um, com uma ponta chanfrada. </li><li> Backfill as pipetas de microinjeção com óleo de parafina utilizando uma seringa de 10 ml, e depois montar a pipeta de microinjeção para um micr homebuiltoinjection aparelho (Figura 2). NOTA: O aparelho de injecção de óleo hidráulico homebuilt consiste de um motor da grade controlado por um interruptor de pedal. Um interruptor adicional muda o modo de viragem do motor. Este motor acciona um parafuso micrômetro (0,5 mm / volta) que avança ou retrai o êmbolo de uma seringa de vidro de 10 mL. A injecção é a uma velocidade de cerca de 3 rpm e uma volta de 0,5 corresponde a uma injecção de nL 50 em um folículo. A ponta da seringa está ligada ao tubo de politetrafluoretileno de paredes espessas que está ele próprio ligado à agulha de injecção por um período muito curto de tubagem Tygon peça. O capilar de injecção é mantida por um mandril que é controlada por um micromanipulador. O sistema inteiro é preenchido com óleo de parafina. As bolhas de ar devem ser evitados, uma vez que irá prejudicar o sistema de injeção. A configuração é iluminada com uma fonte de luz fria. Um estereomicroscópio é necessário para controlo óptico. Folículos são visualizados sob luz fria com um stereomicroscope (40X). Desempenho da instalação de injecção é testado pela injecção de marcador radioactivo em oócitos de Xenopus. Um volume de 45 - 55 nL deve ser administrada por injecção. </li><li> Com uma pipeta com uma ponta de plástico estéril, testar a configuração, colocando uma gota de água estéril para o interior limpo de um termoplástico resistente à umidade (2 x 2 cm). Imergir a ponta da pipeta de injecção para esta gota, e, em seguida, ligar o motor para retrair o êmbolo (pressão negativa no interior da pipeta de injecção). NOTA: A água deve inserir a pipeta e formam uma interface com o óleo visível. </li><li> Retrair a ponta da pipeta a partir da gotícula e aplicar pressão positiva para o interior da pipeta de injecção. Uma gota de água deve formar-se a ponta da pipeta de injecção. </li><li> Preparar para a injecção dos folículos, alinhando-as com os pólos vegetais que aponta para cima nos interstícios de uma malha de nylon (G: 0,8 mm) coladas à parte inferior de um Peprato de tri (60 mm) coberto com MBS. </li><li> Dispensar 2 mL de mRNA usando uma pipeta com uma ponta de plástico estéril para o interior de um termoplástico limpo. Tome-se o ARNm para a pipeta de injecção por aplicação de pressão negativa para o interior da pipeta de injecção. </li><li> Posicionar a pipeta de injeção mais de um folículo individual usando o micromanipulador. </li><li> Inserir a agulha de injecção no centro do pólo vegetal e injectar 50 nL de ARNm a uma taxa de fluxo de 0,6 mL / min através da aplicação de pressão positiva para o interior da pipeta. Espere 5 - 10 s antes de remover a ponta de injecção pipeta a partir do folículo para evitar mRNA fuga. NOTA: O ARNm deve ser injectado no pólo vegetal (amarelado) para evitar a injecção para dentro do núcleo, que está localizado no hemisfério animal. </li><li> Transferir os folículos injetados para uma nova placa de Petri (35 mm) carregada com 2 mL de MBS. Coloque o prato em um refrigerador de vinho fixada em 18 ° C. Incubar os folículos injetados para 1 -7 d antes da gravação, em função da identidade da proteína expressa recentemente. </li></ol> 3. decapagem dos folículos (Figura 3) NOTA: Como mencionado acima, o oócito coberta por uma camada vitelino, as células do folículo e do tecido conjuntivo, o qual contém os vasos sanguíneos 24, é conhecido como um &quot;folículo.&quot; Todas as camadas, excepto para a camada de vitelina, que proporciona estabilidade mecânica sem impedir o acesso das soluções para a superfície da célula, deve ser removido antes de experiências electrofisiológicas. O folículo, sem as camadas de células circundantes tenha sido previamente denominado um oócito &quot;arruinado&quot; 25. Este passo é normalmente realizada no mesmo dia que as experiências electrofisiológicas. <ol><li> Transferência de dez folículos injectado para um tubo de vidro de borosilicato contendo 0,5 mL de MBS, 1 mg / mL de colagenase, e 0,1 mg / mL de inibidor de tripsina. Mergulha-se o tubo num banho de água mantido a 36 ° C. incubar afolículos durante 20 minutos e agitar os tubos ocasionalmente. </li><li> Lavar os folículos, transferindo-os para um segundo tubo contendo 1 ml de MBS à TA. Deixá-los no tubo durante cerca de 10 s. </li><li> Transferir os folículos com um terceiro tubo contendo 0,5 ml de MBS duplamente concentrada contendo 4 mM de etilenoglicol-bis (2-aminoetiléter) - N, N, N &#39;, N&#39;-tetra-acético (EGTA), e incubar durante 4 min a RT. Agitar o tubo de vez em quando. NOTA: A solução hipertónica (MBS duplamente concentrada) é utilizado para induzir a retracção dos oócitos dentro de camadas de células do folículo, destacando-se, assim, as camadas de células foliculares do oócito; EGTA é adicionado para inibir a actividade da colagenase. </li><li> Lavar os folículos, transferindo-os para um segundo tubo contendo 1 ml de MBS à TA. Deixá-los no tubo durante cerca de 10 s. </li><li> Transferir os oócitos para uma placa de Petri (35 mm de diâmetro) contendo 2 mL de MBS. Sob um microscópio estereoscópico, separar os envelopes exteriores dos folículos porbasta apertar o oócito nua distância utilizando o aro de platina. NOTA: Os envelopes exteriores dos oócitos manter firmemente a placa de cultura e podem ser facilmente separados dos oócitos. Alguns ovócitos espontaneamente perder seus envelopes exteriores e será recuperado como oócitos desnudados. Este procedimento resulta em uma preparação relativamente estável, que mantém a aparência esférica dos oócitos. </li></ol> 4. Rápido Solução Mudança em torno do ovócito <ol><li> Realizar um experimento de fixação de voltagem, como descrito 9,26. </li><li> Mudar a solução de perfusão do sistema alimentado por gravidade, rodando os interruptores de perfusão tal como exigido pela experiência. <ol><li> Aplicar a solução de perfusão de 6 ml / min através de um capilar de vidro com um diâmetro interno de 1,35 mm, a boca do qual é colocado cerca de 0,4 mm a partir da superfície do oócito, para permitir trocas rápidas na concentração de agonista em torno do oócito. NOTA: A taxa de variação foi anteriormente estimativad como 70% em menos de 0,5 s 27. </li></ol></li></ol>

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The authors have nothing to disclose.

Os métodos descritos neste artigo desviar os utilizados tradicionalmente 7-9. É padrão para expor os lóbulos do ovário para uma 1 a 2 h colagenase tratamento 8; isolar intactas, oócitos desnudados; e injectar-los com mRNA usando dispositivos de injecção comerciais. Este procedimento clássico tem os seguintes inconvenientes: 1) Os oócitos são susceptíveis de ser danificadas pela longa exposição a altas concentrações de colagenase. 2) Os oócitos desnudados instáveis ​​deve ser a...

Oócitos de Xenopus são amplamente utilizados como um sistema de expressão (Referências: 2 - 3, e suas referências). Eles são capazes de montar correctamente e incorporar proteínas de múltiplas subunidades funcionalmente activos nas suas membranas plasmáticas. Usando este sistema, é possível investigar funcionalmente proteínas da membrana por si só ou em combinação com outras proteínas, a fim de estudar as propriedades de mutantes, ou proteínas quiméricas concatenados, e para pesquisar potenciais drogas. Vantagens da utilização de oócitos em relação a outros sistemas de expressão heterólogos incluem o manuseamento simples das células gigantes, a elevada proporção de células que expressam a informação genética estrangeira, o controlo simples do ambiente do oócito por meio de perfusão de banho, e o controlo do potencial de membrana . O inconveniente deste sistema de expressão é a variação sazonal observado em muitos laboratórios 17-20. A razão para esta variaçãoestá longe de ser clara. Além disso, a qualidade dos oócitos é frequentemente observado de variar fortemente. Os métodos tradicionais 7-9 incluíram o isolamento de lóbulos de ovário, a exposição dos lóbulos de ovário de colagenase para algumas horas, a selecção de oócitos desnudados, e a microinjecção de oócitos. Aqui, uma série de procedimentos alternativos, rápidos são informou que nos permitiram trabalhar com este sistema de expressão por mais de 30 anos sem variação sazonal e pouca variação na qualidade do oócito. Os métodos melhorados e modificados descritos aqui para o isolamento de oócitos, microinjecção com ARNc, e a remoção de camadas de células foliculares podem ser utilizados para a expressão de qualquer proteína de escolha no oócito de Xenopus. O método muito simples para modificações solução rápida do meio em volta do oócito pode ser aplicada ao estudo de qualquer receptor ionotrópico e dos transportadores.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

A oócitos de Xenopus como um sistema de expressão heteróloga de proteínas, foi primeiramente descrito por Gurdon et ai. 1 e tem sido amplamente utilizadas desde a sua descoberta (Referências: 2 - 3, e suas referências). Uma característica que faz com que o oócito atraente para expressão do canal estrangeira é o pobre abundância de canais de iões endógenos 4. Este sistema de expressão tem se mostrado útil para a caracterização de muitas proteínas, entre eles, canais iónicos dependentes de ligandos. A expressão de receptores de GABAA em oócitos de Xenopus e sua caracterização funcional é aqui descrita, incluindo o isolamento de oócitos, micro-injecções com ARNc, a remoção das camadas de células foliculares, e alterações solução rápida em experiências electrofisiológicas. Os procedimentos foram otimizados neste laboratório 5,6 e desviar os utilizados rotineiramente 7-9. Tradicionalmente, os oócitos são desnudados preparadocom um tratamento prolongado colagenase de lóbulos de ovário à TA, e estes são desnudados oócitos microinjectados com ARNm. Usando os métodos optimizados, proteínas de membrana diferentes foram expressos e estudada com este sistema, tais como os receptores recombinantes GABA A, os canais de cloreto 10-12 recombinantes humanos de tripanosomas, 13 canais de potássio 14, e um transportador 15 de mio-inositol, 16. Os métodos descritos aqui podem ser aplicados para a expressão de qualquer proteína de escolha em oócitos de Xenopus, e a mudança rápida solução pode ser utilizada para estudar outros canais iónicos dependentes de ligandos.

Oócitos de Xenopus foram mecanicamente escolhido usando uma ansa de platina (Figura 1). Os oócitos foram micro-injectados com ARNm que codifica para as subunidades GABA A do receptor a 4, P 2, δ, 0,5: 0,5: 2,5 fmol / oócito (Figura 2). Após 4 d, camadas de células foliculares foram removidos (Figura 3). Os oócitos foram voltagem fixada em -80 mV e expostos a concentrações cres...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus Oócitos: Métodos otimizados para microinjeção, remoção de camadas de células foliculares e rápidas mudanças Solução em experimentos eletrofisiológicos

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

xenopus-oocytes-optimized-methods-for-microinjection-removal

Research

JoVE Journal

Biology

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K visualizações.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

Vídeo: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulação e<em&gt; In Vitro</em&gt; Maturação<em&gt; Xenopus laevis</em&gt; Oócitos, seguido de injeção intracitoplasmática de espermatozóide, para estudar o desenvolvimento embrionário

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Biologia do Desenvolvimento

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Braçadeira de tensão Fluorometria em<em&gt; Xenopus</em&gt; Oócitos Usando Aminoácidos Fluorescentes Não-naturais

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Bioquímica

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonagem Funcional Utilizando um<em&gt; Xenopus</em&gt; Oocyte Sistema de Expressão

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Manipulação funcional do gene materno produtos Usando<em&gt; In Vitro</em&gt; Maturação do oócito em Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjeção de Oócitos Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biologia

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizando Localização RNA em Xenopus Oócitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Fertilização de Oócitos Xenopus Utilizando o método de transferência do host

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

Ablação a Laser Multifóton de Centros Organizadores de Microtúbulos Citoplasmáticos em Oócitos de Camundongos

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Avaliação do mouse microinjeção de oócitos, maturação e ploidia

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis como um modelo para identificar Tradução Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Xenopus Oócitos: Métodos otimizados para microinjeção, remoção de camadas de células foliculares e rápidas mudanças Solução em experimentos eletrofisiológicos

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