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.

<ol>
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The authors have nothing to disclose.

Nous ici en détail une nouvelle méthode pour épuiser les facteurs maternels ou surexpression des facteurs exogènes avant la fécondation. Ce système exige deux micro-injections, mais au lieu saute l&#39;opération de grenouilles, utilisé dans le procédé de transfert de 7,17 hôte. Il est optimal pour supprimer l&#39;étape de la chirurgie de la grenouille en termes de soins aux animaux. En outre, nous ne avons pas à prendre en compte pour la qualité de l&#39;hôte femmes grenouilles pour les expériences de transfert, ce qui signifie que nous pouvons nous débarrasser d&#39;un facteur biologique qui affecte le succès d&#39;expériences. Par conséquent, dans ce système, la qualité des ovocytes obtenus à partir de grenouilles PMSG amorcées est un facteur biologique majeur qui est la clé pour les expériences réussies. La qualité des ovocytes peut être jugé après la collecte des defolliculation ovaire ou après. Si aucune anomalie ne est observée lors de ces étapes, il est optimal pour recueillir un autre ovaire d&#39;une nouvelle grenouille. Un autre point quand vous pouvez vérifier la qualité de l&#39;ovocyte est après la maturation des ovocytes. Si moins de 80% de la progestéroneovocytes e-traités montrent des signes de maturation, vous ne pouvez pas être en mesure d&#39;obtenir le nombre suffisant d&#39;embryons pour d&#39;autres analyses. L&#39;utilisation de defolliculation enzymatique lieu de defolliculation manuel est également un autre avantage de l&#39;utilisation de ce système afin de réduire le travail nécessaire pour defolliculation. Cependant, il est encore possible que defolliculation manuel peut donner un meilleur développement que le traitement enzymatique. Comme le montre la figure 3, près de 10% des ovocytes in vitro mûri peut atteindre le stade de têtard piscine. Ces données sont recueillies auprès de 13 expériences indépendantes, y compris ceux présentés dans huit et de nouvelles expériences d&#39;injection. méthode de transfert de l&#39;hôte a besoin de 75 à 150 ovocytes dans chaque traitement pour obtenir des données significatives depuis 30-60% des ovocytes transférés peut atteindre le stade neurula 17. Notre méthode commence normalement par 200 à 300 ovocytes dans chaque groupe expérimental depuis environ 40-60% des embryons clivés peut atteindre lestade de la réponse musculaire. Ces données suggèrent que les deux méthodes prennent en charge un taux de développement raisonnable. Nous avons jusqu&#39;ici obtenu 10 grenouilles vivant de contrôle ARNm injecté et le contrôle antisens ovocytes oligo-injecté à l&#39;aide de cette technique, indiquant que cette approche favorise le développement par la métamorphose. Notre méthode de manipulation des ovocytes-ICSI fournit l&#39;occasion de tester le rôle des facteurs maternels pendant le développement embryonnaire très tôt, dès après la fécondation. En outre, la surexpression de facteurs modificateurs de la chromatine avant la fécondation peut permettre de remodeler la chromatine maternelle avant la fécondation pour comprendre États chromatine maternelle nécessaire pour le développement. Cette stratégie peut aussi bien travailler avec les systèmes d&#39;édition de gènes actuels tels que la transcription activateur comme effecteur nucléase (TALEN) 18,19 CRISPR et / CAS9 20,21 puisque ceux-ci peuvent être exprimées avant même la fécondation. Par conséquent, notre nouvelle approche a un potential à être utilisé pour de nombreuses applications à l&#39;avenir.

Xenopus laevis est largement utilisé, puissant organisme modèle pour étudier le développement 1. Ce est parce que les œufs de Xenopus sont inhabituellement élevé (environ 1/2 à 1/4 mm, par rapport à leurs homologues de mammifère étant d&#39;environ 0,1 mm) et abondante. Les œufs contiennent des composants mère synthétisés et stockés, qui sont suffisants pour conduire le développement embryonnaire jusqu&#39;à la mi-blastula transition (MBT, commise au stade 8-8,5 avec 4,000-8,000 cellules). MBT est accompagné par l&#39;activation du génome zygotique, qui produit alors des produits de gènes embryonnaires qui dirigent le développement. De nombreuses études visant à identifier les facteurs maternels importants pour le développement. De nombreuses études se appuient sur ​​l&#39;injection d&#39;oligonucléotides antisens (oligos), y compris des oligonucléotides morpholino dans embryons fécondés, dans lesquels la dégradation des protéines maternels de cas peut être observé au stade de la gastrula 2-4. Alternativement, les ARNm sont injectés pour em fécondébryons de perturber les fonctions des gènes ou de retracer destins de protéines surexprimés. Cependant, l&#39;injection dans les embryons au stade une cellule-normalement ne affecte pas les niveaux de protéines d&#39;expression maternelle à des stades embryonnaires précoces très avant MBT. Heasman et Wylie établi la méthode de transfert de l&#39;hôte de surmonter ce problème 5. Dans leur méthode, défolliculés manuellement ovocytes sont injectés avec des oligos antisens et transférés dans des femelles hôtes 6. Les protéines sont régulés à la baisse avant la fécondation afin que les rôles de protéines réprimés dans le développement embryonnaire précoce peuvent être examinées. Cette méthode de déplétion maternelle a conduit à l&#39;identification de plusieurs rôles de développement uniques de protéines maternelle, tel que révisé à 7. Dans ce rapport, nous détaillons notre méthode développée récemment, dans lequel l&#39;épuisement maternel ou la surexpression de l&#39;ARNm avant la fécondation est réalisée sans defolliculation manuel et le transfert de l&#39;hôte8. Defolliculation manuel nécessite beaucoup de temps et le transfert de l&#39;hôte doit souvent techniques habiles et la licence spécifique pour la chirurgie des animaux, entravant ainsi l&#39;usage fréquent de la méthode de l&#39;épuisement maternel. Defolliculation et le transfert de l&#39;hôte sont substitués par defolliculation enzymatique 9,10 et injection intracytoplasmique de spermatozoïdes (ICSI) 11-13, respectivement. ICSI aux œufs a été utilisé pour produire des grenouilles transgéniques 12. Sperme et embryons noyaux ont également été transplantés dans des ovocytes in vitro amphibiens mûri 11,14,15. Ici, nous montrons notre méthode étape par étape pour injecter des spermatozoïdes in vitro ovocytes maturés qui ont été pré-injecté avec oligos antisens ou 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.

Le développement embryonnaire des embryons ICSI utilisant dans des ovocytes in vitro mûri a été examiné (figure 3A). taux d&#39;ovocytes GV à la phase de maturation MII sont variables et dépendent en grande partie sur la qualité de l&#39;ovocyte. Dans de bonnes expériences, presque 100% des ovocytes GV répondre à la progestérone et montrent des signes de maturation ovocytaire, devenant finalement des œufs au stade MII. Tous les ovocytes ont été soumis à mûri ICSI et environ 25% des œufs injectés clivé (figure 3A, n = 13 pour le contrôle brouillés ovocytes oligo-injectée et n = 7 pour aucun des ovocytes oligo-injectée). Environ 60% ou 80% des embryons clivés, produites à partir d&#39;ovocytes oligo-injecté contrôle ou ovocyte non-injecté, respectivement, ont atteint le stade blastula / gastrula. Parmi les embryons blastula / gastrula, environ la moitié des embryons dans des échantillons d&#39;oligo-injecté étaient de bonne qualité (presque aucun signe de division anormale et l&#39;apoptose), tandis que 82% des embryons blastula / gastrula étaient de bonne qualité nsur-injection des échantillons (figure 3A). Enfin, 41% et 11% des embryons clivés dans des échantillons d&#39;oligo-injecté a atteint la réponse musculaire natation et les étapes de têtards, respectivement, tandis que 60% ​​et 29% des embryons clivés dans des échantillons non injectée ont fait (figure 3A). Ces embryons ICSI sont le mélange d&#39;embryons normaux et anormaux (figure 3B). Certains d&#39;entre eux subissent une métamorphose et développer à mûrir grenouilles 8. Ces résultats suggèrent que l&#39;injection d&#39;oligonucléotides antisens dans des ovocytes de GV, suivis par l&#39;IVM et ICSI, permet le développement embryonnaire précoce efficace. Bien que l&#39;injection de antisens se oligos diminue développement embryonnaire, nous sommes toujours en mesure d&#39;obtenir suffisamment d&#39;embryons pour de nombreuses fins expérimentales telles que la vérification des taux de développement, RT-PCR, western blot et ainsi de suite. <img alt="Figure 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Figue<html> ure 1: Des exemples typiques de Xenopus laevis ovocytes de bonne qualité ou de mauvaise qualité pour des expériences de maturation in vitro. (A) Un exemple de mauvaises ovocytes de qualité. Par exemple, les ovocytes montrant la pigmentation inégale ne sont pas utilisés pour des expériences ultérieures. Chaque ovocyte de Xenopus laevis est d&#39;environ 1.2 à 1.4 mm de diamètre. (B) Une partie de l&#39;ovocyte défolliculés. La moitié droite de l&#39;ovocyte est recouverte par les cellules de follicules, ce qui peut être perçue que par la présence de vaisseaux sanguins. Une flèche indique une zone exempte de cellules folliculaires et dans lequel l&#39;aiguille d&#39;injection est injecté. (C) Un exemple de bonnes ovocytes de qualité, qui sont de même taille et montrent hémisphères animales uniformément pigmentés avec un contraste évident entre l&#39;hémisphère animal et de l&#39;hémisphère végétatif. 2496 / 52496fig2highres.jpg &quot;/&gt; Figure 2:. Xenopus laevis ovocytes avant et après la maturation Après la maturation des ovocytes, des taches blanches claires apparaissent en haut des hémisphères animales et les cellules folliculaires sont décollée. Chaque ovocyte de Xenopus laevis est d&#39;environ 1 mm de diamètre. <img alt="Figure 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Figure 3: Développement d&#39;embryons ICSI, produites à partir dans des ovocytes in vitro mûri. (A) le développement d&#39;embryons ICSI à chaque étape est résumée. Les ovocytes ont été injectés avec contrôle brouillé oligonucléotides antisens (injection d&#39;oligo de contrôle) ou sans injection (non injection), suivis par l&#39;IVM et ICSI. Le nombre correspondant d&#39;embryons à chaque étape est indiqué ci-dessus les barres. Moyenne ± SEM est indiquée. N = 4-13 expériences indépendantes / f différenteemales. (b) des exemples de survie des embryons ICSI. Ces embryons au stade de bourgeon caudal ont été produits dans une expérience, dans laquelle environ 200 ovocytes ont été utilisés en tant que matériau de départ.

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.

Manipulation et<em&gt; In Vitro</em&gt; Maturation du<em&gt; Xenopus laevis</em&gt; ovocytes, Suivi par injection intracytoplasmique de spermatozoïdes, pour étudier le développement embryonnaire

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 vues.  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.

Vidéo: 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.

Utilisation de l&#39;analyse confocale<em&gt; Xenopus laevis</em&gt; Chargé d&#39;enquêter sur les modulateurs de Wnt et Shh morphogène Dégradés

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

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Biologie du développement

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.

Stem Cell-like<em&gt; Xenopus</em&gt; Embryonnaires explants à étudier les caractéristiques du développement précoce Neural<em&gt; In Vitro</em&gt; Et<em&gt; In 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.

Clonage fonctionnelle en utilisant un<em&gt; Xenopus</em&gt; Système d&#39;expression de l&#39;ovocyte

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.

Technique à la cible microinjection du Developing<em&gt; Xenopus</em&gt; 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 ...

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.

Manipulation fonctionnelle du gène maternel Produits utilisant<em&gt; In vitro</em&gt; Ovocyte Maturation en Zebrafish

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.

Visualisation et analyse quantitative de l&#39;angiogenèse embryonnaire dans<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.

Microinjection d&#39;ovocytes de Xenopus laevis

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

Biologie

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 fertilisation des Ovocytes de Xenopus En utilisant la méthode de transfert d&#39;hôte

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.

Imagerie des cellules vivantes et de l&#39;analyse quantitative des cellules embryonnaires épithéliales 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 production de transgéniques Xenopus laevis Intégration par médiation des enzymes de restriction et de la transplantation nucléaire

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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Chapitres dans cette vidéo

Utilisation de l'analyse confocale

Stem Cell-like

Clonage fonctionnelle en utilisant un

Technique à la cible microinjection du Developing

Manipulation fonctionnelle du gène maternel Produits utilisant

Visualisation et analyse quantitative de l'angiogenèse embryonnaire dans

Microinjection d'ovocytes de Xenopus laevis

La fertilisation des Ovocytes de Xenopus En utilisant la méthode de transfert d'hôte

Imagerie des cellules vivantes et de l'analyse quantitative des cellules embryonnaires épithéliales Xenopus laevis

La production de transgéniques Xenopus laevis Intégration par médiation des enzymes de restriction et de la transplantation nucléaire