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.

Wir hier ausführlich eine neue Methode, um mütterliche Faktoren zum Abbau oder exogene Faktoren vor der Befruchtung überexprimieren. Dieses System erfordert zwei Mikroinjektionen, sondern überspringt die Operation von Fröschen, in der Host-Übertragungsmethode 7,17 verwendet. Es ist optimal, um den Frosch Operation Schritt im Hinblick auf die Pflege der Tiere zu entfernen. Darüber hinaus haben wir nicht zu berücksichtigen, für die Qualität der Gast weiblichen Frösche für die Transferexperimente zu nehmen, was bedeutet, dass wir von einem biologischen Faktor, der den Erfolg der Experimente betrifft loszuwerden. Daher in diesem System die Qualität der Eizellen von PMSG grundiert Frösche erhalten ist ein wichtiger Faktor, der biologischen Schlüssel für erfolgreiche Experimente ist. Die Qualität der Eizellen nach der Entnahme der Eierstöcke oder nach defolliculation beurteilt werden. Wenn irgendeine Abnormalität in diesen Phasen beobachtet wird, ist es optimal, einen weiteren Ovar von neuen Frosches sammeln. Ein weiterer Punkt, wenn man die Qualität der Oozyten zu überprüfen, ist nach Eizellreifung. Wenn weniger als 80% der progesteronE-behandelten Oozyten zeigen Anzeichen von Reife, können Sie nicht in der Lage, die genug Anzahl von Embryonen für weitere Analysen zu erhalten. Die Verwendung von enzymatischen defolliculation statt manuell defolliculation ist auch ein weiterer Vorteil der Verwendung dieses Systems, um die Arbeit für defolliculation reduzieren. Jedoch ist es immer noch möglich, dass eine manuelle defolliculation kann eine besser als der enzymatischen Behandlung. Wie in 3 gezeigt, fast 10% der in vitro gereiften Eizellen können die Schwimm Kaulquappenstadium erreichen. Diese Daten werden aus 13 unabhängigen Experimenten einschließlich der in 8 und neue Injektion Versuche berichtet gesammelt. Host-Übertragungsmethode benötigt 75-150 Oozyten in jeder Behandlung zur Gewinnung von aussagekräftigen Daten, da 30-60% der übertragenen Oozyten können die Neurulastadium 17 zu erreichen. Unsere Methode beginnt normalerweise mit 200-300 Oozyten in jeder Versuchsgruppe seit etwa 40-60% der gespalten Embryonen können die zu erreichenMuskelreaktion Bühne. Diese Daten legen nahe, dass beide Methoden unterstützt eine angemessene Entwicklungsrate. Bisher haben wir erhalten 10 Leben Frösche von Steuer mRNA injiziert und Kontrollantisense-Oligo-injizierten Oozyten mit dieser Technik, was darauf hinweist, dass dieser Ansatz unterstützt die Entwicklung durch Metamorphose. Unsere Oozyte manipulations ICSI Verfahren bietet die Möglichkeit, die Rolle der mütterliche Faktoren in sehr frühen embryonalen Entwicklung zu testen, kurz nach der Befruchtung. Darüber hinaus kann die Überexpression von Chromatin modifizierenden Faktoren vor der Befruchtung ermöglichen es, mütterlichen Chromatin vor der Befruchtung für das Verständnis für die Entwicklung notwendige mütterliche Chromatinzustände umzugestalten. Diese Strategie kann auch gut mit aktuellen Gen-Editing-Systeme wie TALENs (TALEN) 18,19 und CRISPR / CAS9 20,21 da diese schon vor der Befruchtung ausgedrückt werden zu arbeiten. Daher hat unser neuer Ansatz eine potential, für viele Anwendungen in der Zukunft verwendet werden.

Xenopus laevis ist eine weit verbreitete, leistungsfähige Modellorganismus, um die Entwicklung 1 zu untersuchen. Dies liegt daran, Xenopus Eiern ungewöhnlich groß sind (ungefähr 1,2-1,4 mm, im Vergleich zu Säugetier-Gegenstücke etwa 0,1 mm) und reichlich vorhanden. Eier enthalten mütterlich synthetisiert und gespeichert Komponenten, die genug, um die embryonale Entwicklung bis Mitte Blastula Übergang fahren sind (MBT, in der Phase 8-8,5 auftretenden mit 4.000-8.000 Zellen). MBT wird durch zygotischen Genom-Aktivierung, die dann produziert embryonalen Genprodukten Weiterentwicklung direkt verbunden. Zahlreiche Studien zielte darauf ab, mütterliche Faktoren wichtig für die Entwicklung zu identifizieren. Viele Untersuchungen beruhen auf der Injektion von Antisense-Oligonukleotide (Oligos) mit Morpholino-Oligonukleotide in befruchtete Embryonen, wobei Abbau der mütterlichen Proteine ​​können im Gastrula-Stadium 2-4 einzuhalten. Alternativ werden mRNAs befruchteten em injiziertbryonen um Genfunktionen zu stören oder zu Schicksale überexprimiert Proteine ​​zu verfolgen. Jedoch ist die Injektion in die Ein-Zell Embryonen normalerweise nicht beeinflusst Expressionsniveaus von mütterlichen Proteine ​​bei den sehr frühen embryonalen Stadien vor MBT. Heasman und Wylie gründete die Host-Übertragungsverfahren, um dieses Problem zu überwinden, 5. In ihrer Methode werden manuell defollikuliert Oozyten, die mit Antisense-Oligos injiziert und in Wirts Weibchen 6 übertragen. Proteine ​​werden vor der Befruchtung herunterreguliert, so dass die Rollen von regulierte Proteine ​​in frühen embryonalen Entwicklung untersucht werden können. Das mütterliche Abreicherungsverfahren führte zur Identifizierung von mehreren einzigartigen Entwicklungsrollen mütterlicher Proteine, wie in 7 überprüft. In diesem Bericht werden wir ausführlich unsere neu entwickelte Methode, bei der mütterliche Erschöpfung oder Überexpression von mRNA vor der Befruchtung wird ohne manuelle defolliculation und Host-Übertragungs erreicht8. Hand defolliculation erfordert viel Zeit und Host-Übertragungs braucht oft geschickte Techniken und die spezifische Lizenz für Tierchirurgie, wodurch die häufige Verwendung von mütterlichem Erschöpfung Verfahren behindern. Defolliculation und Host-Übertragungs durch enzymatische defolliculation 9,10 und intrazytoplasmatische Spermieninjektion (ICSI) von 11 bis 13 ersetzt sind. ICSI, Eier wurde ursprünglich zur transgenen Fröschen 12 zu produzieren. Sperma und embryonalen Kerne wurden in in vitro gereiften Eizellen Amphibien 11,14,15 transplantiert. Hier zeigen wir unsere Schritt-für-Schritt-Methode, um Spermien in vitro gereiften Eizellen, die waren zu injizieren mit Antisense-Oligos oder pre-mRNA injiziert.

<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.

Embryonalentwicklung ICSI-Embryonen unter Verwendung von in vitro gereiften Eizellen wurde (3A) untersucht. Reifungsgeschwindigkeiten von GV-Oozyten, um den MII Stufe sind variabel und hängt weitgehend von der Qualität der Oozyten. In guten Experimente fast 100% der GV-Oozyten reagieren auf Progesteron und zeigen Anzeichen von Eizellreifung, zuletzt als Eier am MII Stadium. Alle gereiften Eizellen wurden ICSI (7 ohne Oligo-injizierten Oozyten 3A, n = 13 für die Steuerung verwürfelten oligo-injizierten Oocyten und n =) unterzogen und etwa 25% der injizierten Eier abgespalten. Etwa 60% oder 80% des gespaltenen Embryonen aus Steuer oligo-injizierten Oozyten oder nicht injizierten Oozyten erzeugt jeweils erreichte die Blastula / Gastrulastadium. Unter den Blastula / Gastrula Embryonen waren von guter Qualität (fast keine Anzeichen von anomaler Spaltung und Apoptose) etwa die Hälfte der Embryonen in Oligo-Proben injiziert, während 82% der Blastula / Gastrula Embryonen waren von guter Qualität in non-injizierten Proben (3A). Schließlich, 41% und 11% der gespaltenen Embryonen in Oligo-injizierten Proben erreichten die Muskelreaktionen und die Schwimm Kaulquappe Stufen eingesetzt werden, wobei 60% und 29% der gespaltenen Embryonen nicht injizierten Proben war (3A). Diese ICSI-Embryonen werden die Mischung aus normalen und abnormalen Embryonen (3B). Einige von ihnen Metamorphose und zu entwickeln, um Frösche 8 reifen. Diese Ergebnisse legen nahe, dass die Injektion von Antisense-Oligonukleotide in GV-Oozyten, gefolgt von IVM und ICSI, ermöglicht eine effiziente frühen Embryonalentwicklung. Obwohl die Injektion von Antisense-Oligos selbst abnimmt Embryonalentwicklung, sind wir immer noch in der Lage, genug Embryonen für viele experimentelle Zwecke wie etwa die Überprüfung Entwicklungsraten zu erhalten, RT-PCR, Western-Blot und so weiter. <img alt="Figur 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Feige<html> ure 1: Typische Beispiele für Xenopus laevis Oozyten von guter Qualität oder schlechte Qualität für in-vitro-Reifung Experimente. (A) Ein Beispiel für eine schlechte Qualität Oozyten. Beispielsweise werden Oozyten zeigt fleckige Pigmentierung nicht für nachfolgende Experimente verwendet. Jede Xenopus laevis etwa 1,2-1,4 mm im Durchmesser. (B) Ein teilweise defollikuliert Eizelle. Die rechte Hälfte der Eizelle durch Follikelzellen, die durch das Vorhandensein der Blutgefäße erkennen kann abgedeckt. Ein Pfeil zeigt einen Bereich frei von Follikel-Zellen und in der die Injektionsnadel injiziert wird. (C) Ein Beispiel für gute Qualität Oozyten, die gleich große sind und zeigen, gleichmäßig pigmentierte Tierhalbkugeln mit klaren Kontrast zwischen dem Tier Hemisphäre und der pflanzlichen Hemisphäre. 2496 / 52496fig2highres.jpg &quot;/&gt; Abbildung 2:. Xenopus laevis Oozyten vor und nach der Reifung Nach Eizellreifung, an der Spitze der Tierhälften und Follikelzellen erscheinen klare weiße Flecken sind abgezogen. Jede Xenopus laevis Oozyten ist etwa 1 mm im Durchmesser. <img alt="Figur 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Abbildung 3: Entwicklung der ICSI-Embryonen aus in vitro gereiften Eizellen produziert. (A) Entwicklung von ICSI-Embryonen auf jeder Stufe zusammengefasst. Oocyten injiziert mit Steuer verwürfelten Antisense-Oligonukleotide (Oligo Steuereinspritzung) oder ohne Injektion (non-Injektion), gefolgt von IVM und ICSI. Die entsprechende Anzahl von Embryonen in jeder Stufe oberhalb der Balken dargestellt. Mittelwert ± SEM dargestellt. N = 4-13 unabhängigen Experimenten / verschiedene females. (B) Beispiele für überlebende ICSI-Embryonen. Diese Schwanzknospen-Embryonen wurden in einem Experiment, bei dem etwa 200 Oozyten wurden als Ausgangsmaterial verwendet wird produziert.

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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 und<em&gt; In-vitro-</em&gt; Ausbau der<em&gt; Xenopus laevis</em&gt; Eizellen, von Intrazytoplasmatische Spermieninjektion gefolgt, um embryonale Entwicklungsstudie

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

22.4K Aufrufe.  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.

Serie: 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.

Mittels konfokaler Analyse<em&gt; Xenopus laevis</em&gt; Um Modulatoren der Wnt und Shh Morphogen Gradienten Untersuchen

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

Forschung

Entwicklungsbiologie

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.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<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.

Functional Cloning Mit ein<em&gt; Xenopus</em&gt; Eizellen 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 ...

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.

Technik auf Zielmikroinjektions an die Entwicklungs<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.

Funktionelle Manipulation der mütterlichen Gene Produkte verwenden<em&gt; In Vitro</em&gt; Oozytenreifung in Zebrabärbling

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.

Visualisierung und quantitative Analyse der embryonalen Angiogenese in<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.

Mikroinjektion von Xenopus laevis Oozyten

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.

Die Befruchtung der Xenopus Oozyten Über den Host-Transfer-Methode

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.

Live-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in 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.

Herstellung transgener Xenopus laevis Durch Restriktionsenzym-vermittelte Integration und Reaktorsicherheit 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 ...

Manipulation und

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