Este trabalho foi financiado pelo Ministerio de Educación y espanhol Ciencia (BIO 2006-11792) e da Generalitat de Catalunya (2005-SGR00437). Autores gostariam de agradecer Marc Puigcerver e Jonatan Lucas para a sua assistência técnica na preparação dos meios de comunicação. Todo o pessoal da d&#39;Servei Estabulari é reconhecido por alojamento dos animais e, especialmente, Juan Jose Martin e Javier Benito para a sua contribuição adicional sobre a gravação parte deste vídeo. BCN é um companheiro da Fundação Português parágrafo a Ciência e Tecnologia e SG é um companheiro do espanhol Ministerio de Educación y Ciencia.

Cuidados com os animais e os procedimentos foram realizados de acordo com as diretrizes e as normas aprovadas pelo Comitê de Ética em Pesquisa Animal e Humana da Universitat Autònoma de Barcelona, ​​Espanha. I. coleta de oócitos <ol><li> Verifique e selecione hamsters feminino (Mesocricetus auratus; 12/08 semana de idade) da cepa de Ouro da Síria para a presença de um corrimento vaginal espesso (descarga pós-ovulatória). </li><li> Induzir fêmeas selecionadas para superovulate por injeção intraperitoneal de 40 UI de PMSG (Pregnant Mare Serum Gonadotropin), seguido de 40 UI de hCG (gonadotropina coriônica humana) 60 horas depois. </li><li> Euthanize hamsters fêmeas 16 horas após a administração de hCG em uma câmara de dióxido de carbono. </li><li> Isolar ovidutos das fêmeas, como mostrado no vídeo e transferi-los para uma queda de KSOM-H médio. </li><li> Coletar oócitos cumulus complexos rasgando a ampola do oviduto sob um microscópio estereoscópico em uma gota de hialuronidase (156 U / ml) a 37 ° C. Oócitos serão liberados a partir de células cumulus em cerca de 5 minutos. </li><li> Pick up ovócitos de hialuronidase e lavá-los duas vezes em KSOM-H médio. </li></ol> Para a coleta de embriões de camundongos, ratos fêmeas (Mus musculus; 6-8 semana de idade) são induzidos a superovulate por injeção intraperitoneal de 5 UI de PMSG seguido por 5 UI de hCG 48 horas mais tarde e acasaladas com ratos machos. As fêmeas são sacrificadas por deslocamento cervical em 24-26, 44-46 ou 54-56 horas após a administração de hCG para obtenção de pronuclear, 2 de células ou embriões de 4 células estágio, respectivamente. Colheita de embriões pronuclear é realizado exatamente como descrito para oócitos de hamster. Embriões nas fases 2 e 4 células de células são recolhidas através de lavagem com o ovidutos KSOM-H médio [6]. Nota: Na sequência deste protocolo aproximadamente 20 a 30 oócitos / embriões podem ser coletadas de cada fêmea. II. Preparação de soluções de congelamento e descongelamento <ol><li> Prepare solução que eu diluindo 0,57 ml de propilenglycol em 5 ml de KSOM-H (1,5 M de propilenglycol). </li><li> Prepare a solução de descongelamento, adicionando 2 ml de KSOM-H para um tubo com 0,2052 g de sacarose (0,3 M de sacarose). </li><li> Preparar a solução de congelamento, adicionando 2 ml de solução I a um tubo com 0,0684 g de sacarose (1,5 M de sacarose e 0,1 propilenglycol M). </li></ol> Nota: Eu Solution e congelamento e descongelamento, as soluções devem ser preparadas imediatamente antes de seu uso. No entanto, as quantidades especificadas de sacarose a ser usada para a preparação do congelamento e descongelamento soluções podem ser ponderados com antecedência e armazenado em 3 ml de tubos em temperatura ambiente por vários meses. III. Protocolo de congelamento <ol><li> Ovócitos transferência / embriões a partir do meio KSOM-H para uma gota de solução I e incubar por 7-15 minutos em temperatura ambiente. </li><li> Enquanto isso, prepare uma placa de Petri 90 milímetros para carregar a palha, colocando 90 gotas mL de congelamento e descongelamento soluções como é mostrado no vídeo (1 gota de cada solução / palha). </li><li> Transferir o número desejado de oócitos / embriões para as gotas de solução de congelamento. Nota: Nós normalmente transferência entre 5 a 30 oócitos ou embriões / gota. </li><li> Anexar uma palha para um sistema de aspiração de boca controlada e carregá-lo seguindo esta ordem: solução I, do ar, queda de congelamento com os oócitos, ar, queda de descongelamento, o ar. </li><li> Uma vez que a palha é carregada, manter aspiração até que a primeira gota introduzido (solução I) atinge o polímero no topo do canudo e sela o fim. Termo-selar o outro lado da palha ou selar com um plugue de plástico. </li><li> Ligue o freezer programável e selecione o programa de congelamento. O programa de congelamento utilizado neste protocolo é baseado nas taxas de resfriamento publicado originalmente por Lassalle et al. [7]: <ul><li> 2 ° C / min da temperatura ambiente até -7 ° C 0 ° C / min para 10 min para permitir equilíbrio de temperatura e indução de semeadura (veja abaixo) </li><li> 0,3 ° C / min de -7 ° C a -30 ° C 0 ° C / min por 2 min para permitir equilíbrio de temperatura </li><li> 35 ° C / min de -30 ° C a -130 ° C </li></ul></li><li> Carregar os canudos nos slots do titular do congelador programável, certificando-se que a parte da palha com a gota de solução descongelamento é voltado para fora. Inicie o programa de congelamento. </li><li> Quando a temperatura da amostra atinge -7 ° C ea refrigeração está em espera, executar semeadura manual: mergulhar a pinça em nitrogênio líquido, puxe os titulares um pouco fora do freezer para expor a parte da palha contendo as gotas de solução de descongelamento, e imediatamente toque nesta parte do canudos com o fórceps. Repita a operação para cada titular e cada um de palha no suporte, e deixar que o programa de congelamento de correr até o fim. </li><li> Quando o programa estiver concluído, mergulhe os titulares em nitrogênio líquido. </li><li> Retire as palhas datitulares e transferi-los para um gobelet rotulados. </li><li> Finalmente, guarde o gobelet dentro de um tanque de nitrogênio líquido. </li></ol> IV. Descongelamento protocolo <ol><li> Remover a palha do tanque de nitrogênio líquido. </li><li> Manter a palha em temperatura ambiente por 40 segundos. </li><li> Mergulhe a palha em banho-maria a 30 ° C durante 40 segundos. </li><li> Retire a ficha da palha ou cortar a ponta termo-selado. </li><li> Esvaziar a palha numa placa de Petri de 35 mm e misture delicadamente as duas gotas que contêm o congelamento e descongelamento soluções. </li><li> Deixe oócitos / embriões na mistura por 15 minutos em temperatura ambiente. </li><li> Transferência de oócitos / embriões frescos para uma queda de KSOM-H para um adicional de 15 minutos a 37 ° C. </li><li> Oócitos / embriões estão agora prontos para manipulação posterior. </li></ol> Nota: Durante as etapas 6 e 7 você verá que os oócitos descongelados / embriões lentamente inchar devido à reidratação. Representante Resultados V. O uso do protocolo de criopreservação aqui relatados resulta em uma taxa de sobrevivência de cerca de 95% para oócitos de hamster e de&gt; 90%, 85% e 80% para mouse B6CBAF1 embriões no pronuclear, 2-estágios e de célula de 4 células, respectivamente. Quando embriões de camundongos são cultivadas in vitro em KSOM após o descongelamento, pelo menos 70 a 80% devem chegar ao estágio de blastocisto em condições de cultura ideal. É recomendado que você incluir um grupo controle (palha) de oócitos / embriões em cada experimento de congelamento e descongelamento que este grupo antes do resto dos oócitos / embriões são usados ​​para manipulação posterior. Se as taxas de sobrevivência para o grupo controle é menor do que o esperado, descongele outra palha do mesmo lote. Se as taxas de sobrevivência para este segundo grupo também são baixos, descarte as palhetas restantes deste lote de congelamento.

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

Com este protocolo de oócitos e embriões hamster rato pode ser criopreservados com sucesso. Uma vez congelados, os oócitos / embriões podem ser armazenados em tanques de nitrogênio líquido por tempo indeterminado e recuperado a qualquer momento ou local desejado. Isto oferece a vantagem de ter amostras quase pronto para uso sempre que necessário. Por outro lado, este protocolo também pode ser usado para gerar cryobanks embrião de linhagens de camundongos valiosos (como linhagens transgênicas), como uma alternativa econômica e seg...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French type mini-straw 0.25 ml</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>13407/0010</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Farma-Lepori, Spain</td>
<td>749036.4</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-3884</td>
<td>&nbsp;</td>
<tr>
<td>KSOM-H medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref. [11]</td>
</tr>
<tr>
<td>Liquid Nitrogen </td>
<td>&nbsp;</td>
<td>Air Liquide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic plugs</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>19046</td>
<td>&nbsp;</td>
<tr>
<td>PMSG</td>
<td>&nbsp;</td>
<td>Intervet, Spain</td>
<td>1.614/8447</td>
<td>&nbsp;</td>
<tr>
<td>Propilenglycol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>82280</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>107687</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Scissors</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BC-060R; BG-061R</td>
<td>&nbsp;</td>
<tr>
<td>Thermo-sealer</td>
<td>&nbsp;</td>
<td>SIZ</td>
<td>220</td>
<td>&nbsp;</td>
<tr>
<td>35 and 90 mm culture dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>153066; 150350</td>
<td>&nbsp;</td>
<tr>
<td>10 ml tubes</td>
<td>&nbsp;</td>
<td>Greiner Bio-one</td>
<td>163160</td>
<td>&nbsp;</td>
<tr>
<td>Nitrogen tank</td>
<td>&nbsp;</td>
<td>MVE, Cryogenics</td>
<td>XC 43/28</td>
<td>&nbsp;</td>
<tr>
<td>Programmable Freezer</td>
<td>&nbsp;</td>
<td>Planner Products Ltd.</td>
<td>Kryo-10 Series III</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BD-331R; BD-053R; OC-020R</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

collection and cryopreservation of hamster oocytes and mouse embryos

Embriões e oócitos criopreservados com sucesso pela primeira vez mais de 30 anos atrás, quando Whittingham<i&gt; Et al.</i&gt;<sup&gt; 1</sup&gt; E Wilmut<sup&gt; 2</sup&gt; Separadamente descrito que embriões de camundongos poderiam ser congeladas e armazenadas a -196 ° C e, alguns anos mais tarde, Parkening<i&gt; Et al</i&gt;.<sup&gt; 3</sup&gt; Relatou o nascimento de filhotes vivos resultantes de fertilização in vitro (FIV) de oócitos criopreservados. Desde então, o uso de técnicas de criopreservação difundiu-se rapidamente para se tornar um componente essencial da prática de humanos e animais de reprodução assistida e na conservação dos recursos genéticos animais. Atualmente, existem dois principais métodos utilizados para criopreservar oócitos e embriões: congelamento lento e vitrificação. Uma grande variedade de abordagens têm sido usadas para tentar melhorar as técnicas e milhões de animais e milhares de crianças que nasceram a partir de embriões criopreservados. No entanto, subsistem deficiências significativas associadas à criopreservação ainda precisam ser superadas, uma vez que a formação de cristais de gelo, os efeitos da solução e choque osmótico parecem causar cryoinjuries vários pós-descongelamento de oócitos e embriões. Congelamento lento com freezers programável tem a vantagem de utilizar baixas concentrações de crioprotetores, que são geralmente associados com toxicidade química e choque osmótico, mas a sua capacidade para evitar a formação de cristais de gelo em baixas concentrações é limitado. Congelamento lento também induz efeitos super-resfriamento que deve ser evitado o uso de semeadura manual ou automático<sup&gt; 4</sup&gt;. No processo de vitrificação, altas concentrações de crioprotetores inibir a formação de cristais de gelo e levar à formação de um estado vitrificado vitrificados em que a água se solidifica, mas não expandido. No entanto, devido à toxicidade do cyroprotectants nas concentrações utilizadas, os oócitos / embriões só podem ser expostos à solução crioprotetora por um período muito curto de tempo e em uma solução de volume mínimo, antes de submergir as amostras diretamente em nitrogênio líquido<sup&gt; 5</sup&gt;. Na última década, a vitrificação tem se tornado mais popular porque é um método muito rápido em que nenhum equipamento caro (freezer programável) é necessária. No entanto, o congelamento lento continua a ser o método mais amplamente utilizado para oócito / embrião criopreservação. Neste vídeo-artigo vamos mostrar, passo-a-passo, como coletar e congelar lentamente oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Neste vídeo-artigo apresentamos uma demonstração passo-a-passo sobre como coletar e criopreservar oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

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 ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.8K visualizações.  Universitat Autonoma de Barcelona. Neste vídeo-artigo apresentamos uma demonstração passo-a-passo sobre como coletar e criopreservar oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

Vídeo: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

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

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissecção de 6,5 embriões de camundongos dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

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 ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

In Utero injeção intraventricular e eletroporação de embriões de camundongos E15

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

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 ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Criopreservação de embriões pré-implantatório de bovinos, ovinos e caprinos

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Criopreservação de embriões de camundongos por Vitrificação Etileno Glicol-Based

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservação do mouse Sperm and Recovery utilizando o I · Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

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.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

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 ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Coleção celular única de células trophoblastos em embriões humanos de estágio de peri-implantação

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...