Me gustaría dar las gracias a Mike Dewey del Centro de Genética de archivo Peromyscus por su ayuda, paciencia y conocimiento.

Los ovocitos / embriones de recuperación <ol><li> Al menos dos horas antes de la recuperación de ovocitos / embriones, KSOM se coloca en el 37 º C incubadora de CO 2 y FHM se deben descongelar a partir de -20 º. Un plato de vidrio que contiene tres pozos de poca profundidad cóncava se utiliza a las caídas de la casa de KSOM en la incubadora. Para prevenir la deshidratación, la KSOM y el plato se colocan en un recipiente con tapa que tiene el agua en la parte inferior. Este aparato evita la evaporación sin el uso de aceite mineral. </li><li> Los oviductos de una hembra ovula se colocan en los medios de FHM. Para garantizar una orientación y un infindibulum intacta, una pequeña sección del ovario se corta a lo largo de la sección del oviducto y pequeñas del útero. Un contundente 30 aguja se utiliza para expulsar a los ovocitos / embriones del oviducto mediante la inserción de la aguja de punta roma en el infindibulum. El infindibulum debe ser utilizado para recuperar los embriones desde el tubo de oviducto es demasiado pequeño para una aguja de pasar. En el caso de que el infindibulum se destruye, el oviducto debe ser tajo o embriones expulsados. </li><li> Ovocitos / embriones se encuentran en los medios de FHM, centrándose en el microscopio las gotas de grasa que se han hundido en la parte inferior de la placa de Petri. Los ovocitos / embriones aislamiento se puede lograr a 20x, ya que proporciona una mayor área de visualización de gran aumento. Embriones / ovocitos parecen ronda gotitas de grasa que tienen un claro anillo a su alrededor. Pipetear con la boca, con un vaso tirado capilar, se utiliza para mover ovocitos / embriones de KSOM para la incubación y su posterior manipulación. </li></ol> Procedimiento para la recuperación de los ovocitos Preparación <ol><li> Descongele FHM y preparar KSOM. </li><li> Unas horas antes, KSOM colocarlo en un recipiente la extracción de óvulos y el lugar en el cuadro de la punta con agua en el fondo, lugar en incubadora de CO 2. </li><li> Traiga jaulas para el laboratorio y matar a las hembras de la cosecha. </li><li> Cortar el oviducto, teniendo cuidado de cortar algunos de los ovarios y algunos del útero para asegurarse de que no se cortó parte de oviducto. </li><li> Lugar en los medios de FHM en pequeña placa de Petri. </li><li> Vierta un par de placas de Petri FHM los medios de comunicación que se utilizarán para el lavado. </li></ol> Flushing <ol><li> Use una aguja de lavado (30 calibre romo) conectado a una aguja de 1 ml con final en espiral. </li><li> Tome un oviducto a la vez y con la aguja, insertar en infundíbulo encuentra cerca de la parte del ovario izquierdo adjunto. Si el infundíbulo se destruye y no se puede utilizar, luego tomar dos pinzas y sostener un extremo del oviducto. Con la pinza de otros, apriete suavemente, como un tubo de pasta de dientes, para expulsar el embrión. Nota: Esto creará una gran cantidad de desorden en la placa de Petri, ya que una gran cantidad de tejido se vienen con él, por lo que es posible que desee utilizar otra placa de Petri después. </li><li> Para ver los embriones, se centran en el microscopio de las piezas de grasa y buscar todo &quot;pedazo de grasa&quot; con el círculo claro que lo rodea. Nota: Los embriones tienen el mismo aspecto que la grasa, lo que puede ser difícil de encontrar. Los embriones se hunden hasta el fondo, a fin de buscar allí. </li><li> Hacer frotis vaginal de las hembras receptoras. En esta etapa, deben ser pseudopreñadas. </li></ol>

En este artículo, se demuestra la recuperación de los ovocitos de la ovulación inducida (superovuladas) Peromyscus. El uso de superovulación (en comparación con el ciclo estral natural) es común debido a un número mucho mayor resultado. Sin embargo, se debe tener cuidado al interpretar los resultados de la utilización de ovocitos como / embriones, ya que tanto las hormonas utilizadas para inducir la ovulación y el cultivo puede tener efectos. Cuando estro ha sido confirmado, las mujeres son euthenized y los oviductos se rec...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Flushing needle</td>
<td>Tool</td>
<td>Zephyrtronics</td>
<td>ZT5-130-L</td>
<td>30 gauge blunt</td>
</tr>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5060</td>
<td>&nbsp;</td>
<tr>
<td>Peromyscus</td>
<td>Animal</td>
<td>Peromyscus Genetic Stock Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5880</td>
<td>&nbsp;</td>
<tr>
<td>FHM HEPES Buffered Media</td>
<td>Reagent</td>
<td>Specialty Media</td>
<td>MR-025-d</td>
<td>&nbsp;</td>
<tr>
<td>KSOM Embryo Culture Medium (5x10 ml)</td>
<td>Reagent</td>
<td>Specialty Media</td>
<td>MR-020P-5F</td>
<td>&nbsp;</td>
<tr>
<td>Mouth Pipette</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Pulled from glass capillaries and attached to rubber tubing</td>
</tr>
<tr>
<td>Oocyte holding dish</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Glass container with concave divets</td>
</tr>		</tbody>
		</table>
		</div>

retrieval of mouse oocytes

Hasta la fecha, sólo unos pocos estudios han informado de manipulaciones éxito de la embriogénesis Peromyscus o la biología reproductiva. Junto con el archivo Peromyscus Centro Genético (http://stkctr.biol.sc.edu), estamos caracterizando las diferencias relevantes necesarios para desarrollar este sistema. Un objetivo principal ha sido optimizar los ovocitos / recuperación temprana del embrión.

Watch this Scientific Journal Video about Retrieval of Mouse Oocytes at JoVE.com

Retrieval of Mouse Oocytes

Este protocolo se muestra la técnica para la extracción de los ovocitos o embriones en estadio temprano fertilizados del oviducto de los ratones. La capacidad de identificar la infindibulum e insertar una aguja de punta roma en que es esencial para la correcta realización del procedimiento.

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 ...

Microsoft Bing

Research

JoVE Journal

Biology

27.2K vistas.  University of California, Irvine (UCI). Este protocolo se muestra la técnica para la extracción de los ovocitos o embriones en estadio temprano fertilizados del oviducto de los ratones. La capacidad de identificar la infindibulum e insertar una aguja de punta roma en que es esencial para la correcta realización del procedimiento.

Video: Retrieval of Mouse Oocytes

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

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

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

Microinyección de oocitos de Xenopus laevis

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

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.

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 ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

Primer captura Extensión: Secuencia de recuperación selectiva de las fuentes de ADN muy degradado

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

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.

Visualizar la localización del ARN en Xenopus Los ovocitos

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

Fertilization of Xenopus oocytes using the Host Transfer Method

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

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

La fertilización de Oocitos de Xenopus Utilizando el método de transferencia de acogida

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

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.