This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

Este protocolo foi aprovado pelo Comitê de Cuidado e Uso Institucional Animal (IACUC) da Universidade da Geórgia. 1. Preparativos <ol><li> Para a cultura de oócitos, ou acabado de preparar a compra Meio Essencial Mínimo (MEM) e completar com 3 mg / ml de albumina de soro bovino (BSA) tal como delineado na Tabela 1. Colocar uma garrafa de poliestireno em um equilíbrio de carga (tara a zero). Adicionar todos os reagentes, exceto BSA, em ordem e trazer o volume final com o MQ-água, em peso, para um total de 250 g. Em seguida, adicionar o BSA, para permitir dissolver e esterilizar filtro. Nota: O meio MEM descrito requer equilíbrio e a incubação de células com uma mistura de gases medicinais de 5% de CO 2, 5% de O2 e 90% de N2. No entanto, outros meios de comunicação (por exemplo, CZB) pode ser usado para permitir que a incubação de células em 5% de CO2 sob condições atmosféricas. </li><li> Para microinjeção de oócitos, comprar ou preparar o meio M2. </li><li> Prepare pde soro de gonadotropina de égua reinante (PMSG) a uma concentração de 5 IU / 100 ul. </li><li> Compra e reconstituir estoques de siRNA para uma concentração de trabalho de 1 uM. </li></ol> 2. Rato Oocyte Colecção <ol><li> DIA 1: Para estimular o desenvolvimento folicular ovariano e aumentar o número de folículos pré-ovulatórios, administrar 5 IU de PMSG intraperitoneal a ratos do sexo feminino de 48 horas antes da coleta de oócitos. </li><li> DIA 3: Para a coleta de oócitos e cultura, um aumento de 35 mm placas de cultura com 3 ml de MEM / BSA suplementado com 1 mg / ml milrinona. Este inibidor da fosfodiesterase mantém os oócitos em prophase-I prender e evita rompimento da vesícula germinativa (GVBD). Equilibrar as placas de cultura de uma mistura de gases medicinais para, pelo menos, 15 min. Nota: Milrinona meio suplementado é necessária durante todo coleta de oócitos, bem como a microinjeção de oócitos e a 24 hr cultura hold período pós siRNA. </li><li> Recuperar os ovários de camundongos fêmeas usando laboratório estabelecido pratica 15 e transfira para uma nova cultura prato com pré-aquecido e equilibrada MEM / BSA / milrinona. </li><li> Coloque a placa de cultura no palco de um microscópio estereoscópico para coleta de ovócitos. Datas de cumulus-oócito (COC-complexos de) por punção manualmente os folículos antrais com 27 agulhas G. Fixar um ovário para a parte inferior da placa de cultura, com uma agulha de 27 G ligada a uma seringa de 1 ml, e utilizar uma segunda agulha para perfurar todos os folículos grandes. Repita o procedimento para cada ovário. </li><li> Usando procedimentos boca pipetagem padrão ou outros meios de aspiração de oócitos, recolher todos os oócitos que são cercados por 2 ou mais camadas de células do cumulus compactas. Transferir a COC de novo para um prato e incubar a 37 ° C durante 1 h. </li><li> Remova cuidadosamente os cercam as células do cumulus por pipetagem suave repetido com uma pipeta 1 ml ajustado para um volume de aspiração de 750 mL. Repita o passo de pipetagem cerca de 12 vezese permitir que os oócitos a recuperar durante 5 min. Continuar dessa maneira até que todas ou a maioria das células do cumulus foram removidas. Transferir os oócitos desnudadas para outro prato de cultura e deixar equilibrar a 37 ° C durante 15 min. </li><li> Atribuir os oócitos desnudados em três grupos experimentais a ser utilizado para: (i) microinjecção de siRNAs específicos contra o alvo de interesse, (ii) microinjecção de inespecíficas (controlo) siRNAs, e (iii) um grupo não-injectados de controlo para contabilizar condições de cultura. </li></ol> 3. Oocyte Microinjection <ol><li> Colocação em placas de cultura com 3 ml de meio M2 suplementado com milrinona (1 ug / ml) para microinjecção de oócitos. Prepare pratos de cultura contendo MEM / BSA / milrinone para lavar e cultura subsequente de oócitos injectados. Nota: meio M2 contém tampão HEPES e requer pré-aquecimento a 37 ° C durante 15 minutos, sob condições atmosféricas. MEM exige pré-aquecimento e de equilíbrio utilizando gás medicinal como descreverd acima. </li><li> Prepara-se o sistema de micro-injecção. Coloque a agulha de injeção com 5 ml de solução de siRNA específico 1 ï¿½. Fixe a pipeta de exploração e agulha de injeção para micromanipuladores do sistema de microinjeção e configurar a unidade de injeção com volume e pressão calibrada. </li><li> Coloque uma micro-gota de 200 mL de suporte M2 no interior da tampa de um prato de cultura de 3 cm e adicionar um oócito para configurar e de alinhamento do sistema de micro-injecção. </li><li> Usando o oócito como um guia, ajustar as posições da exploração e de injeção de agulhas. Aplicar pressão negativa para a pipeta de retenção para fixar suavemente o oócito e ajustar a posição da agulha de injecção para o maior diâmetro da célula. Ajustar configurações para permitir a microinjecção de cerca de 10 ul da solução de siRNA em qualquer lugar no citoplasma dos oócitos. <img alt="figura 1" src="/files/ftp_upload/53586/53586fig1.jpg" /> Figura 1. Oocyte microinjeção configurar. ( <strong&gt; A) Imagem representativa de uma micro-gota de M2 meios 200 ul colocado sobre a tampa de uma placa de cultura de 3 cm, com a pipeta de retenção da agulha e de injecção posicionados à esquerda e à direita, respectivamente. (B) Grupo de prophase-I preso oócitos (GV-estágio) antes da microinjeção. <a href="https://www.jove.com/files/ftp_upload/53586/53586fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> </li><li> E volta a tampa para a fase estereomicroscópio e adicionar uma micro-gota de 200 mL de suporte M2 fresco. Adicionar um grupo de oócitos (~ 10) para o micro-gota a pipeta boca, ou métodos alternativos, em seguida, retornar a tampa com micro-gota para a fase de microinjeção. <ol><li> Prossiga para microinject os oócitos individuais. Fixar um ovócito com a pipeta, segurando, inserir-se lentamente a ponta da agulha de injecção para dentro do citoplasma e expulsar o volume de injecção de solução de siRNA. Retraia cuidadosamente a agulha de injeção e move o oócito injectado para uma posição, na parte inferior do micro-gota. Mover os oócitos injectados, sem sucesso, para uma posição no topo do micro-gota. </li><li> Repita o procedimento de microinjeção com cada ovócito. Para manter a viabilidade dos oócitos óptima, microinject apenas um pequeno número de oócitos de cada vez. Assegure-se que este processo não leva mais do que alguns minutos, para evitar mudanças de temperatura e pH no micro-gota. </li></ol></li><li> Aspirar oócitos injectados com sucesso ou mover-se cuidadosamente a tampa com o micro-gota de oócitos injectados com o estereomicroscópio. Transferir os oócitos viáveis ​​injectados por pipetagem boca ou métodos alternativos, para MEM / BSA a forma / milrinona para lavar e equilibrar-se a 37 ° C. </li><li> Usando um outro grupo de oócitos, repita o processo de microinjeção com uma nova agulha de injeção carregado com siRNAs não específicas para o grupo controle. Lavar os ovócitos em MEM / BSA / milrinona e transferência para uma cultura prato separado. </li><li> Cultura todos os grupos deoócitos durante 24 horas em MEM / BSA / milrinona a 37 ° C sob uma atmosfera de gás médico. Nota: O período de cultura &#39;bloco milrinona&#39; é necessário para alvo eficiente esgotamento mRNA / proteína. </li></ol> 4. Oocyte Cultura para maturação meiótica <ol><li> Dia 4: Para cada grupo experimental de oócitos set-up 4 pratos de cultura (35 mm) com 3 ml de MEM / BSA. Suplemento um prato meios por grupo com 10% (alta qualidade) de soro fetal de bovino (FBS) a ser usado para a maturação do oócito. Permitir que todos os pratos de cultura para equilibrar a 37 ° C com mistura de gases medicinais. </li><li> Para libertar a partir de oócitos &#39;bloco milrinona&#39;, sequencialmente lavar os oócitos três vezes em placas contendo MEM / BSA e depois transferir os oócitos para o prato de maturação contendo meio com FBS a 10%. Todos os grupos de cultura de oócitos durante 17 horas a 37 ° C, para permitir a retomada e progressão da meiose. </li><li> Dia 5: Preparar soluções para FIXA oócitoção, incluindo: (i) 4% de paraformaldeído (PFA) em tampão PEM (100 mM PIPES [pH 6,9], MgCl 2 1 mM, EGTA 1 mM) com 0,5% de Triton-X 100, e (ii) de PBS suplementado com 5 % de FBS que vai ser usada para lavar e bloquear os oócitos. Nota: Estas soluções requerem pré-aquecimento a 37 ° C, antes da fixação do oócito. </li><li> Para a fixação do oócito (após a cultura de 17 horas), rapidamente transferir cada grupo experimental de oócitos por pipetagem boca ou métodos alternativos em poços individuais (de um 4-bem prato) contendo 750 ul de pré-aquecido solução PFA a 4% e incubar a 37 ° C durante 1 h. Subsequentemente lavar cada grupo de oócitos de 3 vezes (15 minutos cada) em 750 ul de PBS pré-aquecido contendo 5% de FBS. </li><li> Oócitos de bloco em 200 ul de PBS / FBS a 5% O / N a 4 ° C para minimizar a ligação não específica de anticorpos. </li></ol> 5. Análise de imunofluorescência <ol><li> DIA 6: Nota: A imunocoloração é feito de uma placa multi-poços e o OOCytes são serialmente transferidos para poços sequenciais, contendo as respectivas soluções de anticorpos e de lavagem. 48 ou placas de 96 poços pode ser usado, basta ajustar o volume da solução com base no tamanho bem. Para placas de 96 poços utilizar 200 ul por poço. Os oócitos são transferidos para as diferentes soluções pela seguinte ordem: solução de anticorpo primário - 3 - da solução de lavagem de poços anticorpo secundário - 3 poços de lavagem. Para limitar a exposição à luz a placa pode ser coberto com a folha. Preparar uma solução de PBS / 5% de FBS e usar esta solução para preparar as diluições de anticorpos (por exemplo, de coelho anti-pericentrin (1 / 1.000), de ratinho anti-tubulina (1 / 1.000)). </li><li> Transferir os oócitos fixos de cada grupo experimental para poços individuais contendo 200 uL (ou 100 uL, se é desejada menos volume) da solução de anticorpo primário e cobrir o poço com parafilme. Incubar de acordo com as condições específicas do anticorpo óptimas (por exemplo, 37 ° C durante 1 hora, ou 4 ° CO / N). Nota: O anticorpo primário dilution, bem como o tempo de incubação específico e temperatura exigem testes para determinar as condições ideais para diferentes anticorpos utilizados. </li><li> Após a incubação com o anticorpo primário, os oócitos lavar três vezes em PBS / FBS a 5% (10-15 min cada). </li><li> Transferir os oócitos na solução contendo anticorpos secundários conjugados com fluorescência (por exemplo, 1 / 1.000 a diluição dos anticorpos anti-coelho e anti-murganho conjugados com fluorocromos de diferentes comprimentos de onda). Incubar durante 1 hora a 37 ° C. </li><li> Lavar os oócitos três vezes em PBS / FBS a 5% (10 - 15 min cada). </li><li> Após o passo final de lavagem, transferir os oócitos em uma lâmina de vidro limpa e delicadamente aspirar qualquer solução de lavagem em excesso. Isto vai imobilizar os oócitos na superfície do vidro. Adicionar 8 mL de mídia (contendo DAPI) de montagem e sobrepor cuidadosamente a mídia de montagem com uma 22 mm x 22 milímetros lamela. Abaixe a lamela lentamente para evitar bolhas de interceptação e / ou danificar os ovócitos. Nota: Alternativamente, para manter as propriedades de 3-dimensionais do oócito para análise de microscopia confocal, adicionar um pequeno volume de esferas de vidro de 100 uM (misturado com geleia de petróleo) para os cantos da lâmina de cobertura antes da montagem sobre a lâmina. </li><li> Armazenar as lâminas a 4 ° C, ou prosseguir para a avaliação de progressão meiótica, bem como a análise dos níveis de expressão e localização subcelular da proteína utilizando um microscópio de fluorescência equipado com os filtros necessárias para coincidir com os anticorpos secundários utilizados. </li></ol>

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The authors have nothing to disclose

Embora existam vários métodos para a transferência de ácido nucleico exógeno em células somáticas, tais como a electroporação e a transfecção, microinjecção é o método ideal para a administração de moléculas de ARN em oócitos de rato transcricionalmente quiescentes. O protocolo atual fornece uma abordagem eficaz para in vitro esgotamento de mRNAs específicos que permitem que o teste funcional de diferentes fuso e / ou fatores associados à MTOC em oócitos. Esta abordagem resulta em esgotame...

A meiose é um processo de divisão única que ocorre em gametas (óvulos e espermatozóides) e envolve duas divisões sucessivas sem intervir síntese de DNA para separar cromossomos homólogos e cromátides irmãs durante a meiose-I e meiose-II, respectivamente 1. Erros na segregação dos cromossomos durante a divisão da meiose em oócitos pode resultar em aneuploidia, que é herdado pelo embrião durante a fertilização. Notavelmente, a incidência de aneuploidia em embriões em desenvolvimento aumenta com o avançar da idade materna e é uma causa principal de defeitos congénitos congénitas, bem como a perda da gravidez em mulheres 1,2, assim, uma necessidade importante sublinhar a compreender a base molecular de aneuploidia durante a divisão meiótica . Durante a divisão celular, a segregação cromossômica depende fundamentalmente da montagem do aparelho do fuso microtúbulos e estabelecimento de interações cromossômicas-microtúbulos estáveis ​​para a correta fixação ao eixo opostopólos. É importante ressaltar que a formação do fuso meiótico em ovócitos de mamíferos difere da mitose em células somáticas, e é regulada por centros de organização de microtúbulos únicos (MTOCs) que não possuem centríolos 3,4. Proteínas essenciais necessárias para a nucleação de microtúbulos e organização para localizar MTOCs ovócitos, incluindo γ-tubulina que catalisa a montagem de microtúbulos. Além disso, pericentrin funciona como uma proteína essencial andaime, que se liga e âncoras y-tubulina, bem como de outros factores na MTOCs 5. Notavelmente, os nossos estudos mostram que a depleção de proteínas principais associadas interrompe-MTOC organização fuso meiótico e conduz a erros de segregação cromossoma em oócitos, que não são inteiramente resolvidas pelo posto de montagem do eixo (SAC) 6,7. Portanto, defeitos de estabilidade do fuso, que não provocam detenção meiótica, representam um risco significativo na contribuição para a aneuploidia. Apesar de seu papel essencial na montagem do fuso e organização, prot MTOC oócitocomposição e função ein permanece pouco compreendida. Testando a função de proteínas alvo específicas em oócitos de mamíferos é um desafio, como as células tornam-se transcricionalmente quiescente pouco antes do recomeço da meiose 8,9. Assim, os oócitos pré-ovulatórios dependem lojas de ARNm maternais para retomar a meiose e a apoiar divisão meiótica, bem como as primeiras divisões de clivagem após a fertilização 10,11. A eficácia de interferência de RNA (RNAi) degradação mediada de transcritos de mRNA em oócitos de mamíferos está bem estabelecida e RNAs maternas recrutados para a tradução durante a maturação meiótica são particularmente passível de siRNA alvejando 12-14. Portanto, a microinjecção de RNAs interferentes curtas (siARN) em oócitos fornece uma ferramenta valiosa para esgotar mRNAs alvo para testes funcionais. Aqui, descrevemos métodos para o isolamento de oócitos de camundongos e esgotamento mediado por siRNA de transcri específicopts para testar a função de uma proteína essencial MTOC-associado, pericentrin. Além disso, descreve-se as condições de análise de imunofluorescência para avaliar a formação do fuso meiótica em oócitos.

<table border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>EMD Biosciences</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium (MEM)</td>
			<td></td>
			<td></td>
			<td>*Recipe outlined in Table 1</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>Sigma</td>
			<td>E-7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic Acid, sodium salt&#160;</td>
			<td>Sigma</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G, potassium salt&#160;</td>
			<td>Sigma</td>
			<td>P-7794</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Sigma</td>
			<td>S-9137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Sigma</td>
			<td>G-8540</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt dihydrate&#160;</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>Gibco&#160;</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>Sigma</td>
			<td>M-6895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma</td>
			<td>P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax M2 Media with Hepes</td>
			<td>EMD Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNAs targeting Pericentrin</td>
			<td>Qiagen</td>
			<td>GS18541</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control siRNAs&#160;</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pericentrin (rabbit)</td>
			<td>Covance</td>
			<td>PRB-432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin (mouse)</td>
			<td>Sigma</td>
			<td>T-6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21430</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti -mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (SMZ 800)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Fluorescent Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Micro-injections System</td>
			<td>Eppenforf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulators</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-injection needles (femtotips)</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Holding pipettes (VacuTip)</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm culture dishes</td>
			<td>Corning Life Sciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3367</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm CA Filter System</td>
			<td>Corning Life Sciences</td>
			<td>430768</td>
			<td></td>
		</tr>
	</tbody>
</table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

A microinjecção de siRNAs proporciona uma abordagem eficaz para a degradação de ARNm e proteína subsequente depleção em oócitos, que permite o teste funcional eficiente e altamente específico de diferentes factores alvo in vitro. Subsequentemente, a imunofluorescência é utilizado para a análise de fenótipo específico, bem como para validar a depleção de proteínas em oócitos injectados com siRNA. No exemplo atual, marcação fluorescente de oócitos individuais com DAPI em conjunto com anticorp...

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Aqui, apresentamos um protocolo para esgotamento específico mRNA mediado por siRNA seguido por análise de imunofluorescência para avaliar a formação do fuso meiótico e organização em oócitos de camundongos. Este protocolo é adequado para in vitro esgotamento das transcrições e avaliação funcional dos diferentes fuso e / ou fatores associados à MTOC em oócitos.

Meiotic Avaliação de fuso em oócitos mouse por silenciamento mediado por siRNA

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

meiotic-spindle-assessment-mouse-oocytes-sirna-mediated

Research

JoVE Journal

Biology

11.6K visualizações.  College of Veterinary Medicine, University of Georgia.  Aqui, apresentamos um protocolo para esgotamento específico mRNA mediado por siRNA seguido por análise de imunofluorescência para avaliar a formação do fuso meiótico e organização em oócitos de camundongos. Este protocolo é adequado para in vitro esgotamento das transcrições e avaliação funcional dos diferentes fuso e / ou fatores associados à MTOC em oócitos. 

Vídeo: Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Recuperação de oócitos de camundongos

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Recolha e criopreservação de óvulos e embriões de camundongos Hamster

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificação de Citometria de Fluxo de células de camundongo meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imagens ao vivo de cálcio celular Combinado com siRNA silenciamento gênico mediado Identifica Ca 2 + Canais de vazamento na membrana ER e seus mecanismos de regulação

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium-Mediated Virus-Induced Assay silenciamento gênico Em Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Silenciando a longo prazo de Intersectin-1s nos pulmões do rato por entrega repetida de um siRNA específico via lipossomos catiônicos. Avaliação dos efeitos Knockdown por microscopia eletrônica

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...