Name: manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development
Uploaded: 2015-02-09T15:00:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

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The authors have nothing to disclose.

Nos detalle aquí un nuevo método para agotar factores maternos o para sobreexpresar factores exógenos antes de la fertilización. Este sistema requiere dos microinyecciones, pero en su lugar se salta de la cirugía de las ranas, utilizado en el método de transferencia de 7,17 host. Es óptimo para eliminar el paso de la cirugía rana en términos de cuidado de los animales. Por otra parte, no tenemos que tener en cuenta para la calidad de las ranas hembra de acogida para los experimentos de transferencia, lo que significa que podemos deshacernos de un factor biológico que afecta el éxito de los experimentos. Por lo tanto en este sistema la calidad de los ovocitos obtenidos de ranas cebados con PMSG es un factor biológico importante que es la clave para experimentos exitosos. La calidad de los ovocitos puede ser juzgado después de la recogida de ovario o después defolliculation. Si se observa alguna anomalía en estas etapas, es óptimo para recoger otro ovario de una nueva rana. Otro punto en el que se puede comprobar la calidad de los ovocitos es después de la maduración de ovocitos. Si es menos de 80% de progesteronaovocitos e tratados muestran signos de maduración, es posible que no pueda obtener el número suficiente de embriones para otros análisis. El uso de defolliculation enzimática en lugar de defolliculation manual es también otra ventaja de utilizar este sistema para reducir la mano de obra requerida para defolliculation. Sin embargo, todavía es posible que defolliculation manual puede dar un mejor desarrollo que el tratamiento enzimático. Como se muestra en la Figura 3, casi 10% de los ovocitos madurados in vitro se puede llegar a la etapa de natación renacuajo. Estos datos se recogen de 13 experimentos independientes, incluyendo los reportados en 8 y nuevos experimentos de inyección. Método de transferencia Host necesita 75-150 ovocitos en cada tratamiento para obtener datos significativos desde 30 a 60% de los ovocitos transferidos puede llegar a la etapa neurula 17. Nuestro método normalmente se inicia con 200 a 300 ovocitos en cada grupo experimental ya que aproximadamente el 40-60% de los embriones escindidos puede llegar a laetapa respuesta muscular. Estos datos sugieren que ambos métodos apoyan una tasa de desarrollo razonable. Hasta ahora hemos obtenido 10 ranas vivas de control de mRNA inyectado y control antisentido ovocitos oligo-inyectado utilizando esta técnica, lo que indica que este enfoque apoya el desarrollo a través de la metamorfosis. Nuestro método de manipulación-ICSI de ovocitos ofrece la oportunidad de probar el papel de los factores maternos durante el desarrollo embrionario temprano, poco después de la fertilización. Además, la sobreexpresión de factores de modificación de la cromatina antes de la fertilización puede hacer posible para remodelar la cromatina materna antes de la fertilización para la comprensión de los estados de la cromatina materna necesario para el desarrollo. Esta estrategia también puede trabajar bien con los sistemas de edición gen de actualidad como la transcripción del tipo activador de efector nucleasa (TALEN) 18,19 y CRISPR / CAS9 20,21 ya que éstos pueden expresarse incluso antes de la fertilización. Por lo tanto, nuestro nuevo enfoque tiene un potential que debe utilizarse para muchas aplicaciones en el futuro.

Xenopus laevis es un ampliamente utilizado, poderoso organismo modelo para estudiar el desarrollo 1. Esto es porque los huevos de Xenopus son inusualmente grande (aproximadamente 1.2 a 1.4 mm, en comparación con sus homólogos de mamíferos ser aproximadamente 0,1 mm) y abundante. Los huevos contienen maternalmente sintetizadas y almacenadas componentes, que son suficientes para impulsar el desarrollo embrionario hasta la transición a mediados de blástula (MBT, que se producen en la etapa de 8-8,5 con 4.000-8.000 células). MBT es acompañada por la activación del genoma cigóticos, que entonces produce productos de genes embrionarios que dirigen el desarrollo adicional. Numerosos estudios dirigidos a identificar los factores maternos importantes para el desarrollo. Muchos estudios se basan en la inyección de oligonucleótidos antisentido (oligos) incluyendo oligonucleótidos morfolino en embriones fertilizados, en los que la degradación caso de las proteínas maternas se puede observar en la etapa de gástrula 2-4. Alternativamente, los ARNm se inyectan a em fertilizadobryos que perturban las funciones de genes o para rastrear destinos de proteínas sobreexpresados. Sin embargo, la inyección en los embriones en estadio de una célula que normalmente no afecta a los niveles de expresión de proteínas materna en las primeras etapas embrionarias antes de MBT. Heasman y Wylie establecieron el método de transferencia de acogida para superar este problema 5. En su método, los ovocitos desfolicularon manualmente son inyectados con oligonucleótidos antisentido y se transfirieron a hembras de acogida 6. Las proteínas están regulados a la baja antes de la fertilización para que funciones de las proteínas downregulated en el desarrollo embrionario temprano pueden ser examinados. Este método agotamiento de la madre llevó a la identificación de varias funciones de desarrollo únicas de proteínas maternas, tal como fue revisado en 7. En este informe, detallamos nuestro método recientemente desarrollado, en el que se alcanza el agotamiento materno o sobreexpresión de ARNm antes de la fertilización sin defolliculation manual y transferencia de acogida8. Defolliculation manual requiere una gran cantidad de tiempo y de transferencia de acogida a menudo necesita técnicas de expertos y la licencia específica para la cirugía animal, lo que dificulta el uso frecuente de método de agotamiento de la madre. Defolliculation y transferencia de acogida son sustituidos por defolliculation enzimática 9,10 y la inyección intracitoplasmática de espermatozoides (ICSI) 11-13, respectivamente. ICSI a los huevos se utilizó originalmente para producir ranas transgénicos 12. Esperma y embriones de núcleos también fueron trasplantadas en in vitro de ovocitos de anfibios madurado 11,14,15. Aquí, mostramos nuestro método paso a paso para inyectar espermatozoides ovocitos madurados in vitro que fueron pre-inyectados con oligonucleótidos antisentido o ARNm.

<table><tbody><tr><td>Pregnant Mare Serum Gonadotropin (PMSG)&amp;nbsp;</td><td>MSD Animal Health</td><td>Vm 01708/4309</td><td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td><td>Sigma</td><td>A5040</td><td>For anesthesia</td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>For oocyte defolliculation. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Drummond Nanoject</td><td>Drummond Scientific Company</td><td>3-000-205/206</td><td>For microinjection</td></tr><tr><td>glass capillary&amp;nbsp;</td><td>Alpha laboratories</td><td>7&amp;rdquo; Drummond #3-000-203-G/XL</td><td>For microinjection</td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>SUTTER Instrument</td><td>Model D-97</td><td>For microinjection</td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-500</td><td>For &lt;em&gt;in vitro&lt;/em&gt; maturation and ICSI</td></tr><tr><td>14 ml ultra-clear centrifuge tube&amp;nbsp;</td><td>Beckman Coulter United Kingdom Ltd</td><td>344060</td><td>For sperm purification</td></tr><tr><td>OptiPrep Density Gradient Medium (Iodixanol)</td><td>Sigma</td><td>D1556</td><td>For sperm purification</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Cell permeabilization reagent</td></tr><tr><td>Shaker</td><td>Hybaid&amp;nbsp;</td><td>HB-SHK-1</td><td>For oocyte defolliculation</td></tr><tr><td>Dissecting microscope (Stereo zoom microscope)</td><td>ZEISS</td><td>Semi SV6</td><td>For oocyte collection and microinjection</td></tr><tr><td>50 &amp;mu;m pore filter</td><td>CellTrics</td><td>04-0042-2317</td><td>For sperm purification</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100XP</td><td>For sperm purification</td></tr><tr><td>50 ml centrifuge tube</td><td>Cellstar Greiner Bio-One</td><td>227261 or 210261</td><td>Oocyte collection and defolliculation reaction</td></tr><tr><td>15 ml centrifuge tube</td><td>FALCON</td><td>352097</td><td>For sperm purification</td></tr><tr><td>90 mm Petri dish&amp;nbsp;</td><td>Thermo Scientific</td><td>101VR20/C</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 60&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 35&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>351008</td><td></td></tr></tbody></table>

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

El desarrollo embrionario de embriones ICSI utilizando en ovocitos madurados in vitro fue examinado (Figura 3A). Tasas de maduración de ovocitos GV a la etapa MII son variables y dependen en gran medida de la calidad de los ovocitos. En los buenos experimentos, casi el 100% de los ovocitos GV responder a la progesterona y muestran signos de maduración de ovocitos, finalmente convertirse huevos en la etapa MII. Todos los ovocitos madurados fueron sometidos a ICSI y aproximadamente 25% de los huevos inyectados escindidos (Figura 3A, n = 13 para el control revueltos ovocitos oligo-inyectado y n = 7 para no ovocitos oligo-inyectada). Aproximadamente el 60% o 80% de los embriones escindidos, producidos a partir de ovocitos inyectados con oligo de control o de ovocitos no inyectada, respectivamente, alcanzaron la etapa de blástula / gástrula. Entre los embriones blástula / gástrula, aproximadamente la mitad de los embriones en las muestras de oligo-inyectado eran de buena calidad (casi no hay signos de división anormal y apoptosis), mientras que el 82% de los embriones blástula / gástrula eran de buena calidad nmuestras inyectadas en (Figura 3A). Finalmente, 41% y 11% de los embriones escindidos en muestras de oligo-inyectada llegaron a la respuesta muscular y las etapas de natación renacuajo, respectivamente, mientras que el 60% y el 29% de los embriones escindidos en muestras no inyectados hicieron (Figura 3A). Estos embriones ICSI son la mezcla de los embriones normales y anormales (Figura 3B). Algunos de ellos sufren metamorfosis y desarrollar a madurar ranas 8. Estos resultados sugieren que la inyección de oligonucleótidos antisentido en oocitos GV, seguido de IVM y ICSI, permite el desarrollo embrionario temprano eficiente. Aunque la inyección de sí mismo oligos antisentido disminuye el desarrollo embrionario, todavía somos capaces de obtener suficientes embriones para muchos propósitos experimentales como la comprobación de las tasas de desarrollo, RT-PCR, western blot y así sucesivamente. <img alt="Figura 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Higo<html> Ure 1: Ejemplos típicos de Xenopus laevis ovocitos de buena calidad o mala calidad de los experimentos in vitro de maduración. (A) Un ejemplo de ovocitos de mala calidad. Por ejemplo, los ovocitos que muestran la pigmentación irregular no se utilizan para experimentos posteriores. Cada oocito de Xenopus laevis es de aproximadamente 1/2 a 1/4 mm de diámetro. (B) un ovocito parcialmente desfolicularon. La mitad derecha del ovocito está cubierto por las células del folículo, que se pueden discernir por la presencia de vasos sanguíneos. Una flecha muestra un área libre de células del folículo y en la que se inyecta la aguja de inyección. (C) Un ejemplo de ovocitos de buena calidad, que son de igual tamaño y muestran hemisferios animales uniformemente pigmentados con un claro contraste entre el hemisferio animal y el hemisferio vegetal. 2496 / 52496fig2highres.jpg &quot;/&gt; Figura 2:. Oocitos de Xenopus laevis antes y después de la maduración Después de la maduración de ovocitos, manchas blancas claros aparecen en la parte superior de los hemisferios animales y las células del folículo se pelan. Cada oocito de Xenopus laevis es de aproximadamente 1 mm de diámetro. <img alt="Figura 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Figura 3: Desarrollo de embriones ICSI, producidos a partir de ovocitos madurados in vitro. (A) Desarrollo de embriones ICSI a cada etapa se resume. Los oocitos fueron inyectados con control mezclado oligonucleótidos antisentido (oligo de inyección de control) o sin inyección (no inyección), seguido de IVM y ICSI. El número correspondiente de embriones en cada etapa se muestra por encima de las barras. Media ± SEM se muestra. N = 4-13 experimentos independientes / diferente females. (B) Ejemplos de sobrevivir embriones ICSI. Estos embriones tailbud etapa fueron producidos en un experimento, en el que se utilizaron aproximadamente 200 ovocitos como material de partida.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

La manipulación y<em&gt; In Vitro</em&gt; La maduración de<em&gt; Xenopus laevis</em&gt; Los ovocitos, seguido de inyección intracitoplasmática de espermatozoides, para estudiar el desarrollo embrionario

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

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Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

22.5K vistas.  University of Cambridge. We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Video: Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Uso del análisis confocal de<em&gt; Xenopus laevis</em&gt; Encargado de investigar moduladores de Wnt y Shh morfógeno Degradados

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Investigación

Biología del Desarrollo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Celular-como Stem<em&gt; Xenopus</em&gt; Embrionarias explantes para Estudiar Características Temprana del Desarrollo Neural<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonación funcional utilizando una<em&gt; Xenopus</em&gt; Sistema de Expresión de ovocitos

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Técnica de microinyección a Target a los países en desarrollo<em&gt; Xenopus</em&gt; riñón

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

La manipulación de genes maternos funcional de Productos que utilizan<em&gt; In Vitro</em&gt; La maduración de ovocitos en el pez cebra

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualización y análisis cuantitativo de la angiogénesis embrionaria<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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 ...

Biología

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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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Capítulos en este video

Uso del análisis confocal de

Celular-como Stem

Clonación funcional utilizando una

Técnica de microinyección a Target a los países en desarrollo

La manipulación de genes maternos funcional de Productos que utilizan

Visualización y análisis cuantitativo de la angiogénesis embrionaria

Microinyección de oocitos de Xenopus laevis

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

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear