Este trabajo fue apoyado por el Ministerio español de Educación y Ciencia (BIO 2006 hasta 11.792) y la Generalitat de Catalunya (2005-SGR00437). Los autores desean agradecer a Marc Puigcerver y Lucas Jonatan por su asistencia técnica en la preparación de los medios de comunicación. Todo el personal de la Estabulari Servei d&#39;es reconocido por albergar a los animales y, especialmente, Juan José Martín y Javier Benito, por su contribución adicional sobre la grabación de parte de este video. BCN es un miembro de la Fundación un párrafo portugués Ciência e Tecnologia y SG es un compañero de la española Ministerio de Educación y Ciencia.

Cuidado de los animales y los procedimientos se realizaron de acuerdo a los lineamientos y reglamentos aprobados por el Comité de Ética en la investigación animal y humana de la Universitat Autònoma de Barcelona, ​​España. I. recolección de ovocitos <ol><li> Comprobar y seleccionar hamsters hembra (Mesocricetus auratus, 8-12 semanas de edad) de la cepa de oro de Siria a la presencia de una secreción espesa vaginal (flujo postovulatoria). </li><li> Inducir a las mujeres seleccionadas para superovular por inyección intraperitoneal de 40 UI de PMSG (gonadotropina sérica de yegua gestante), seguido de 40 UI de hCG (gonadotropina coriónica humana) 60 horas después. </li><li> Eutanasia hamsters mujer de 16 horas tras la administración de hCG en una cámara de dióxido de carbono. </li><li> Aislar a los oviductos de las hembras, como se muestra en el video y transferirlos a una gota de KSOM-H medio. </li><li> Recoger cúmulos ovocito complejos por romper la ampolla del oviducto bajo un microscopio estereoscópico en una gota de hialuronidasa (156 U / ml) a 37 ° C. Los ovocitos se libera de células del cumulus en aproximadamente 5 minutos. </li><li> Recoger óvulos de hialuronidasa y lavar dos veces en KSOM-H medio. </li></ol> Para la recogida de embriones de ratones, los ratones hembra (Mus musculus; 6-8 semanas de edad) son inducidos a superovular por inyección intraperitoneal de 5 UI de PMSG seguido de 5 UI de hCG 48 horas más tarde y se aparearon con ratones machos. Las hembras son sacrificadas por dislocación cervical en el 24-26, 44-46 o 54-56 horas después de la administración de hCG para la obtención de pronuclear, de 2 o 4 células de células de embriones etapa, respectivamente. Recolección de los embriones pronuclear se lleva a cabo exactamente como se describe en ovocitos de hámster. Embriones en las fases 2-célula y célula-4 son recogidos por el lavado del oviducto con KSOM-H medio [6]. Nota: Después de este protocolo de aproximadamente 20 a 30 ovocitos / embriones se pueden recoger de cada mujer. II. Preparación de congelación y descongelación de soluciones <ol><li> Prepare una solución de I diluyendo 0,57 ml de propilenglicol en 5 ml de KSOM-H (1,5 M de propilenglicol). </li><li> Prepare una solución de descongelación mediante la adición de 2 ml de KSOM-H a un tubo con 0,2052 g de sacarosa (0,3 M de sacarosa). </li><li> Preparar la solución de congelación mediante la adición de 2 ml de solución que a un tubo con 0,0684 g de sacarosa (1,5 M de propilenglicol y 0,1 M de sacarosa). </li></ol> Nota: la solución y la congelación y descongelación, y deben prepararse justo antes de su uso. Sin embargo, las cantidades especificadas de la sacarosa que se utilizará para la preparación de la congelación y descongelación de soluciones puede ser ponderada por adelantado y almacenado en tubos de 3 ml a temperatura ambiente durante varios meses. III. Protocolo de congelación <ol><li> Transferencia de ovocitos / embriones del medio KSOM-H a una gota de la solución I y se incuba durante 7-15 minutos a temperatura ambiente. </li><li> Mientras tanto, prepare una placa de Petri de 90 mm para cargar la paja mediante la colocación de l 90 gotas de congelación y descongelación de soluciones como se muestra en el video (1 gota de cada solución / paja). </li><li> Transferir la cantidad deseada de los ovocitos / embriones de las gotas de solución de congelación. Nota: Por lo general, la transferencia de entre 5 y 30 ovocitos o embriones / drop. </li><li> Adjunte una paja a un sistema de aspiración controlada y la boca de carga es el siguiente orden: solución que yo, el aire, la caída de la congelación de los ovocitos, el aire, la caída de la descongelación, el aire. </li><li> Una vez que la paja se ha cargado, mantener la aspiración hasta que la gota introdujo por primera vez (solución I) alcanza el polímero en el extremo superior de la paja y las juntas este fin. Termo-sellar el otro extremo de la paja o el sello con un tapón de plástico. </li><li> Encienda el congelador programable y seleccione el programa de congelación. El programa de congelación utilizado en este protocolo se basa en la velocidad de enfriamiento, publicada originalmente por Lassalle et al. [7]: <ul><li> 2 ° C / min desde la temperatura ambiente a -7 ° C 0 ° C / min durante 10 minutos para permitir el equilibrio de temperatura y la inducción de la siembra (ver más abajo) </li><li> 0,3 ° C / min de -7 ° C a -30 ° C 0 ° C / min durante 2 minutos para permitir el equilibrio de temperatura </li><li> 35 ° C / min desde -30 ° C a -130 ° C </li></ul></li><li> La carga de la paja en las ranuras de la titular de la nevera programable, asegurándose de que la parte de la paja con la gota de solución de descongelación es hacia el exterior. Iniciar el programa de congelación. </li><li> Cuando la temperatura de la muestra llega a -7 ° C y el enfriamiento está en espera, lleve a cabo la siembra manual: hundir el fórceps en nitrógeno líquido, tire de los titulares de un poco fuera del congelador para exponer la parte de las pajillas que contienen las gotas de la solución de descongelación, y de inmediato tocar esta parte de la paja con las pinzas. Repita este procedimiento para cada titular y la paja en el soporte de cada uno, y luego dejar que el programa de congelación hasta el final. </li><li> Cuando el programa se ha completado, los titulares de la caída en nitrógeno líquido. </li><li> Quitar la paja deltitulares y transferirlos a un gobelet etiquetados. </li><li> Finalmente, coloque la gobelet dentro de un tanque de nitrógeno líquido. </li></ol> IV. Descongelación protocolo <ol><li> Quitar la paja del tanque de nitrógeno líquido. </li><li> Mantener la paja a temperatura ambiente durante 40 segundos. </li><li> Remoje la paja en un baño de agua a 30 ° C durante 40 segundos. </li><li> Retire el tapón de la paja o cortar la punta de la termo-selladas. </li><li> Vaciar la paja en un plato de petri de 35 mm y mezcle suavemente las dos gotas que contienen el congelamiento y descongelamiento soluciones. </li><li> Deja los ovocitos / embriones en la mezcla durante 15 minutos a temperatura ambiente. </li><li> La transferencia de ovocitos / embriones a una caída fresca de KSOM-H por 15 minutos a 37 ° C. </li><li> Los ovocitos / embriones están listos para su posterior manipulación. </li></ol> Nota: En los pasos 6 y 7 que usted debe ver que los ovocitos descongelados / embriones poco a poco se hinchan debido a la rehidratación. V. Los resultados representativos El uso del protocolo de criopreservación que aquí resulta en una tasa de supervivencia de aproximadamente el 95% de los ovocitos de hámster y de&gt; 90%, 85% y 80% para el ratón B6CBAF1 embriones en el pronuclear, dos etapas de las células y de 4 celdas, respectivamente. Cuando los embriones de ratón se cultivaron in vitro en KSOM después de la descongelación, por lo menos 70 a 80% debería llegar a la etapa de blastocisto, en condiciones óptimas de cultivo. Se recomienda que se incluye un grupo de control (paja) de los ovocitos / embriones en cada experimento de congelación y deshielo que este grupo antes que el resto de los ovocitos / embriones se usan para otras manipulaciones. Si las tasas de supervivencia para el grupo control son inferiores a lo esperado, descongelar otra paja del mismo lote. Si las tasas de supervivencia de este segundo grupo son también bajas, desechar la paja que queda de este lote de congelación.

<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>

Con este protocolo de ovocitos de hámster y de embriones de ratón pueden ser criopreservados con éxito. Una vez congelado, los ovocitos / embriones pueden ser almacenados en tanques de nitrógeno líquido por tiempo indefinido y se recuperó en cualquier momento o lugar deseado. Esto ofrece la ventaja de tener muestras de casi listo para su uso cuando sea necesario. Por otro lado, este protocolo también se puede utilizar para generar criobancos embrión a partir de cepas de ratón de gran valor (como las líneas de transgénicos), como ...

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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

Embriones y ovocitos criopreservados fueron los primeros con éxito más de 30 años, cuando Whittingham<i&gt; Et al.</i&gt;<sup&gt; 1</sup&gt; Wilmut y<sup&gt; 2</sup&gt; Descritos separadamente que los embriones de ratón podría ser congelado y almacenado a -196 ° C y, unos años más tarde, Parkening<i&gt; Et al</i&gt;.<sup&gt; 3</sup&gt; Reportó el nacimiento de crías vivas como resultado de la fertilización in vitro (FIV) de ovocitos criopreservados. Desde entonces, el uso de las técnicas de criopreservación se ha extendido rápidamente a convertirse en un componente esencial en la práctica de la reproducción asistida humana y animal y en la conservación de los recursos genéticos de los animales. En la actualidad, existen dos principales métodos utilizados para criopreservar óvulos y embriones: el congelamiento lento y la vitrificación. Una amplia variedad de enfoques se han utilizado para tratar de mejorar tanto las técnicas y los millones de animales y miles de niños han nacido a partir de embriones crioconservados. Sin embargo, subsisten deficiencias importantes asociados a la criopreservación todavía hay que superar, ya que la formación de cristales de hielo, los efectos de la solución y el shock osmótico parecen causar cryoinjuries varios descongelado después de ovocitos y embriones. La congelación lenta, con congelador programable tiene la ventaja de la utilización de bajas concentraciones de crioprotectores, que generalmente están asociados con la toxicidad química y el shock osmótico, pero su capacidad para evitar la formación de cristales de hielo en concentraciones bajas es limitado. La congelación lenta también induce efectos súper-que debe ser evitado el uso de la siembra manual o automático<sup&gt; 4</sup&gt;. En el proceso de vitrificación, las altas concentraciones de crioprotectores inhibir la formación de cristales de hielo y dar lugar a la formación de un estado semejante al vidrio vitrificado en el que el agua se solidifica, pero no ampliado. Sin embargo, debido a la toxicidad de cyroprotectants a las concentraciones utilizadas, los ovocitos / embriones sólo pueden ser expuestos a la solución de crioprotectores por un período muy corto de tiempo y en una solución de volumen mínimo, antes de sumergir las muestras directamente en nitrógeno líquido<sup&gt; 5</sup&gt;. En la última década, la vitrificación se ha vuelto más popular porque es un método muy rápido en el que no se requiere un equipo caro (congelador programable). Sin embargo, la congelación lenta sigue siendo el método más ampliamente utilizado para la criopreservación de ovocitos / embriones. En este video-artículo mostramos, paso a paso, cómo recoger y congelar lentamente ovocitos de hámster con altas tasas de supervivencia después de la descongelación. El mismo procedimiento se puede aplicar para congelar y descongelar con éxito embriones de ratón en las diferentes etapas de desarrollo de preimplantación.

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

En este video-artículo se presenta una demostración paso a paso sobre cómo recoger y criopreservar óvulos de hámster con altas tasas de supervivencia después de la descongelación. El mismo procedimiento se puede aplicar para congelar y descongelar con éxito embriones de ratón en las diferentes etapas de desarrollo de preimplantación.

Recolección y criopreservación de ovocitos de hámster y de embriones de ratón

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 vistas.  Universitat Autonoma de Barcelona. En este video-artículo se presenta una demostración paso a paso sobre cómo recoger y criopreservar óvulos de hámster con altas tasas de supervivencia después de la descongelación. El mismo procedimiento se puede aplicar para congelar y descongelar con éxito embriones de ratón en las diferentes etapas de desarrollo de preimplantación.

Video: 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.

Transferencia nuclear en ovocitos de ratón

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

Investigación

Biología

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]).

La disección de embriones de ratón 6,5 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.

Recuperación de los ovocitos de ratón

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. 

En la inyección intraventricular Utero y electroporación de embriones de ratón 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.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

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.

La criopreservación de embriones de preimplantación de ganado, ovejas y cabras

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.

La criopreservación de embriones de ratón por el etileno a base de glicol vitrificación

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.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

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.

Evaluación del huso meiótico en ovocitos de ratón por silenciamiento siRNA mediada

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.

Colección de células únicas de células trofoblastas en embriones humanos en fase de implantación

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