La disección de los órganos reproductores de una hembra de ratón <ol><li> Los animales en gestación son sacrificados por asfixia de CO 2 de gas comprimido. </li><li> Coloque el animal en decúbito supino almohadilla absorbente y sumergirlo en etanol al 70% para reducir el riesgo de contaminación de la disección con el pelo del ratón. </li><li> Pellizcar la piel y hacer una pequeña incisión lateral en la línea media con regulares tijeras quirúrgicas. Mantener la piel firme por encima y por debajo de la incisión de la piel y tirar hacia fuera de la cabeza y la cola para exponer el abdomen. </li><li> Sujete el peritoneo con una pinza y se corta a exponer la cavidad abdominal. </li><li> Localizar los órganos reproductivos en la región dorsal de la cavidad del cuerpo: dos cuernos uterinos, el oviducto y los ovarios. </li></ol> Raya primitiva etapa (6,5 dpc) Disección <ol><li> Quitar el cuerno uterino, tome el útero por debajo del oviducto y cortar libre a lo largo del mesometrio. Lugar útero disecados en PBS enfriado con hielo. <img src="/files/ftp_upload/old/160_2008_7_8_2/160_figure-1.jpg" alt="Figura 1" width="263" height="132" /> Figura 1 </li><li> Separe cada embrión, por el corte entre los lugares de implantación a lo largo de los cuernos uterinos. <img src="/files/ftp_upload/old/160_2008_7_8_3/160._figure-2.jpg" alt="Figura 2" width="575" height="171" /> Figura 2 </li><li> Sujete el revestimiento del útero muscular deslizando pinzas de relojero entre la capa muscular y el tejido circundante decidua envuelto. Pele la capa muscular, la exposición de la decidua. <img src="/files/ftp_upload/old/160_2008_8_15_4/160_Figure-3.png" alt="Figura 3" width="593" height="166" /> Figura 3 </li><li> Clip de una porción de la decidua expuestos en el vértice (aproximadamente 1 / 5 de los tejidos decidua), que expondrá la punta midventral o distal del embrión encerrado. <img src="/files/ftp_upload/old/160_2008_8_15_5/160_Figure-4.png" alt="Figura 4" width="589" height="212" /> Figura 4 </li><li> El embrión puede ser bombardeado a cabo utilizando las puntas de unas pinzas. Pierce la decidua con unas pinzas que rodea al embrión y pinzas abiertas al desgarro decidua aparte. <img src="/files/ftp_upload/old/160_2008_8_15_6/160_Figure-5.png" alt="Figura 5" width="249" height="200" /> Figura 5 </li><li> Una vez que se extrae de embriones, la membrana Reichart todavía puede estar unido, así como el cono ectoplacental (trofoblasto). El embrión puede ser diseccionado más de una cuidadosa disección. <img src="/files/ftp_upload/old/160_2008_8_15_7/160_Figure-6.png" alt="Figura 6" width="356" height="222" /> Figura 6 </li></ol>

<ol>
	<li>Nagy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: A laboratory manual third edition. , (2003).</li></ol>

Post-implantación en el ratón produce entre 4,5 dpc y el parto. Este método de disección puede ser aplicado, esencialmente como se describe, para aislar antes o después de los embriones en etapa de 5.5-8.5 DPC.

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<td>Micro Dissecting Forceps serrated, straight</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5130 </td>
<td>2 pairs</td>
</tr>
<tr>
<td>Micro Dissecting Tweezers Pattern #55 INOX</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5010 </td>
<td>2 pairs</td>
</tr>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (1X) </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14190 </td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Sterile Polysytrene Petri Dish</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>08-757-12 </td>
<td>&nbsp;</td>
<tr>
<td>4% Paraformaldehyde in PBS</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Straight Sharp-Pointed Dissecting Scissors</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>08-940 </td>
<td>&nbsp;</td>
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<td>Pregnant female mice </td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dissecting Microscope</td>
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dissection of 6.5 dpc mouse embryos

Análisis de patrones de expresión génica durante las primeras etapas del desarrollo embrionario de mamíferos pueden proporcionar pistas importantes sobre la función del gen, la célula-célula la interacción y los mecanismos de señalización que guía los patrones embrionarias. Sin embargo, la disección del embrión de ratón de la decidua, poco después de la implantación puede ser un procedimiento difícil y detalladas paso a paso la documentación de este proceso es insuficiente. Aquí se demuestra cómo después de la implantación (6,5 dpc), los embriones se encuentran aisladas por primera disección del útero de un ratón embarazadas (detección de coitum el tapón vaginal fue designado poist días 0,5) y, posteriormente, la disección del embrión a partir de la decidua materna. La disección de la membrana de Reichert se describe así como la eliminación del cono ectoplacental.

Watch this Scientific Journal Video about Dissection of 6.5 dpc Mouse Embryos at JoVE.com

Dissection of 6.5 dpc Mouse Embryos

El aislamiento de embriones etapa post-implantación, permite estudiar los patrones de genes y analizar células de linaje toma de decisiones durante el desarrollo embrionario, aunque la disección adecuada del embrión temprano puede ser un desafío. Este protocolo describe un método para aislar las primeras etapas primitivas racha de embriones (~ 6,5 días 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 ...

Research

JoVE Journal

Biology

38.0K vistas.  Harvard Medical School. El aislamiento de embriones etapa post-implantación, permite estudiar los patrones de genes y analizar células de linaje toma de decisiones durante el desarrollo embrionario, aunque la disección adecuada del embrión temprano puede ser un desafío. Este protocolo describe un método para aislar las primeras etapas primitivas racha de embriones (~ 6,5 días post coitum [dpc]).

Video: Dissection of 6.5 dpc Mouse Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

La disección del Organizador y explantes Animal Polo de Xenopus laevis embriones y de la Asamblea de un ensayo de adhesión celular

Investigación

Biología

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 ...

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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

La disección de Gyrus dentado del hipocampo de ratones adultos

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

La disección en vivo de Drosophila Los embriones: métodos simplificados para la detección colecciones mutantes tinción con anticuerpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

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 ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Todo el monte In Situ La hibridación de E8.5 a E11.5 embriones de ratón

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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 ...

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR).&#160;This technique allows investigators to study early stages of development that were previously difficult to access.

The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications.

La disección y análisis de aguas abajo de Zebra Finch embriones en etapas tempranas del desarrollo

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...