Name: xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments
Uploaded: 2016-12-31T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

Hayvan deneyleri Bern Kantonu Kantonstierarzt yerel komite, Kantonaler Veterinärdienst Bern (BE85 / 15) tarafından onaylanmıştır. Kara kurbağası cinsi oositlerinde hazırlanması 1. <ol><li> Katı, 20 ° C &#39;de muhafaza edilir, su içinde 12 saat / 12 saatlik aydınlık / karanlık döngüsünde kurbağa (Xenopus laevis) tutun. </li><li> Dişi kurbağa 9&#39;dan yumurtalıkların lobları çıkarın. NOT: lobların kaldırma rejenerasyonu için bir uyarıcı olduğunu ve sık sık oosit kalitesi cerrahisi ile geliştirir. Oosit, bir Vitellin tabakası, folikül hücrelerinin hücre tabakası, ve kan damarları 21 ihtiva eden bağ dokusu ile kaplıdır. tüm yapı &quot;folikülü.&quot; denir </li><li> Yoğun penisilin ve streptomisin (88 mM NaCl, 1 mM KCI, 2.4 mM ile desteklenmiş steril Barth&#39;ın Tuzlu 7 (MBS) içinde oosit içeren folikül ile paketlenir yumurtalıkların lobları yerleştirinNaHCO 3, 0.82 mM MgSO 4, 0.41 mM CaCl2, 0.33 mM Ca (NO 3) 2, 10 mM 4- (2-hidroksietil) -1-piperazinetansülfonik asit; HEPES (pH 7.5, NaOH), 100 ug / ml penisilin ve 100 ug / ml streptomisin). </li><li> Tek çıkış aşaması V-VI 21 folikülleri. forseps ile yumurtalık lob tutun. Köklerinin üzerinde platin döngü (Şekil 1) indirin ve yavaşça yumurtalık dokusundan folikülü içeren bağ dokusu bozmaya döngü çekin. NOT: Sahne V-VI 21 folikül kendi büyüklüğü ile karakterize edilir (1.0 - çapı 1,2 mm) ve koyu pigmente hayvan yarımkürede ve sarımsı bitkisel yarımkürede arasında iyi aksine. </li><li> Bir plastik Pasteur pipeti (1.5 mm&#39;lik bir açıklık çapına geri kesilmiş) vasıtasıyla 35 mm çaplı Petri kabı sadece sağlıklı görünümlü folikülleri (yani mükemmel küresel ve bozulmamış pigmentasyon ile) aktarın. </li></ol> Not: Burada açıklanan mikroenjeksiyon sistemi Kressmann ve Birnstiel 22 tarafından bildirilen türetilmiştir. <ol><li> In vitro transkripsiyon bir alıcı, bir poli (A +) kuyruğu ve RNA miktarının eklenmesi jel elektroforezi 23 tarafından α 4 β 2 δ GABA alt birimleri kodlayan ilgili cDNA&#39;lardan cRNAs hazırlayın. </li><li> Mikropipet çektirmenin kullanarak borosilikat cam kılcal damarlar (1.0 mm dış çapı (OD), 0.58 mm iç çapı (ID), 100 mm uzunluk) den mikroenjeksiyon pipetler hazırlayın. bir eğimli ucu ile 15 mikron - 12 bir ucu çapı oluşturmak için bir mikromanipülatör ve microfilament kullanarak bir mikroskop altında cam kılcal damarların ipuçları kesiyorum. </li><li> 10 ml şırınga kullanılarak parafin yağı ile mikroenjeksiyon pipetler dolgu, ve daha sonra bir homebuilt MICR üzerine mikroenjeksiyon pipet monteoinjection cihaz (Şekil 2). NOT: homebuilt yağ hidrolik enjeksiyon cihazı, bir ayak pedalı ile kontrol edilen bir ızgara motorundan oluşmaktadır. Ek bir anahtar motorun dönme modunu değiştirir. Bu motor, avans ya da 10 uL cam şırınga pistonu geri çekilir bir mikrometre vidası (0,5 mm / dönüş) çalıştırır. Enjeksiyon 3 rpm&#39;lik bir hızda ve bir 0.5 dönüş folikül bir 50 nL enjeksiyon karşılık gelir. şırınga ucu kendisine Tygon hortumunun çok kısa bir parça enjeksiyon iğnesine bağlanır kalın duvarlı politetrafluoroetilen boru bağlanmıştır. Enjeksiyon kılcal bir micromanipulator tarafından denetlenen bir mandrenle tarafından düzenlenmektedir. Tüm sistem parafin yağı ile doldurulur. Onlar enjeksiyon sistemine zarar verecek herhangi bir hava kabarcıkları, kaçınılmalıdır. Kur soğuk ışık kaynağı ile aydınlatılmış. Bir stereomikroskop optik kontrol için gereklidir. Foliküllerinin bir stereomicr soğuk ışık altında görüntülenmiştirOscope (40X büyütme). Enjeksiyon kurulum Performans Xenopus oosit içine radyoaktif izleyici enjeksiyonu ile test edilir. 45 A hacim - 55 nL enjeksiyon başına teslim edilmelidir. </li><li> steril plastik uçlu bir pipet kullanılarak, neme karşı dirençli bir termoplastik (2 x 2 cm), temiz iç kısmına steril bir su damlasının yerleştirilmesi kurulumu test. Bu damlacık içine enjeksiyon pipet ucu batırın ve sonra pistonu (enjeksiyon pipet içinde negatif basınç) geri çekmek için motoru çevirin. NOT: Su pipet girmek ve yağ ile görünür bir arayüz oluşturmalıdır. </li><li> damlacık gelen pipet ucu geri çekin ve enjeksiyon pipet içine pozitif basınç uygulayın. Bir su damlacığı enjeksiyon pipetine ucunda oluşturmalıdır. </li><li> Bir Pe altına yapıştırılmış: bitkisel kutuplar naylon örgü boşlukları (0.8 mm G) yukarı bakacak şekilde onları sıraya folikül enjeksiyon için hazırlayınMBS ile kaplı üç çanak (60 mm). </li><li> Bir termoplastik temiz iç kısmına steril plastik uçlu bir pipet kullanılarak mRNA 2 ul koyun. enjeksiyon pipetine içine negatif basınç uygulayarak, enjeksiyon pipetine mRNA al. </li><li> Mikromanipülatör kullanarak bireysel folikül üzerinde enjeksiyon pipet yerleştirin. </li><li> Bitkisel kutup merkezine iğne takın ve pipet içine pozitif basınç uygulayarak 0.6 mL / dk&#39;lık bir akış oranında mRNA 50 nL enjekte edilir. mRNA kaçış önlemek için folikül enjeksiyon pipet çıkarmadan önce 10 sn - 5 bekleyin. NOT: mRNA hayvan yarımkürede yer alan çekirdeğin içine enjeksiyon önlemek için bitkisel (sarımsı) kutupta enjekte edilmelidir. </li><li> MBS 2 mL dolu yeni bir petri (35 mm) enjekte folikülleri aktarın. 18 ° C&#39;ye ayarlanmış bir şarap soğutucuya çanak yerleştirin. 1 enjekte folikülleri inkübe -7 gün Kayıttan önce, yeni Sentezlenen proteinin ne olduğu ile ilgili olarak. </li></ol> 3. Foliküllerinden soyma (Şekil 3) NOT: olarak bilinen, yukarıda belirtilen kan damarları 24 içeren bir vitelline tabakası, folikül hücreleri ve bağ dokusu ile kaplıdır oosit olarak &quot;folikül.&quot; hücre yüzeyine çözümleri erişim engellemeden, mekanik stabilite temin vitelline tabakası hariç tüm tabakalar elektrofizyolojik deneyler önce çıkarılmalıdır. Çevredeki hücre katmanları olmadan folikül önce bir &quot;soyulmuş&quot; oosit 25 olarak adlandırılmıştır. Bu adım genellikle elektrofizyolojik deneyler ile aynı gün yapılır. <ol><li> Aktarım on MBS 0.5 mL, 1 mg / ml kollajenaz, ve 0.1 mg / ml tripsin inhibitörü içeren bir borosilikat cam tüp köklerinin enjekte edilmiştir. 36 ° C &#39;de muhafaza edilen bir su banyosu içinde tüp bırakın. tasarlamak20 dakika folikülleri ve bazen tüpler sallayın. </li><li> RT&#39;de MBS 1 mL içeren ikinci bir tüp onlara aktararak folikülleri durulayın. yaklaşık 10 saniye süreyle tüp onları bırakın. </li><li> 4 mM etilen glikol-bis (2-aminoetileter) ihtiva eden iki kat konsantre edilmiş MBS 0.5 mL ihtiva eden bir üçüncü tüpe köklerinin transfer - N, N, N &#39;, N&#39;-tetraasetik asit (EGTA) ve 4 dk için inkübe RT. bazen tüp sallayın. Not: hipertonik çözeltisi (iki kat konsantre edildi MBS) ve böylece oositten foliküler hücre katmanları ayırma folikül hücresi tabakaları içinde oosit, bir çekme indüklemek için kullanılır; EGTA kolajenaz aktivitesini inhibe eklenir. </li><li> RT&#39;de MBS 1 mL içeren ikinci bir tüp onlara aktararak folikülleri durulayın. yaklaşık 10 saniye süreyle tüp onları bırakın. </li><li> MBS 2 mL içeren bir Petri kabı (35 mm çap) oosit aktarın. Bir stereomikroskop altında, tarafından köklerinin gelen dış zarfları ayrısadece platin döngü kullanarak Çıplak oosit iterek. NOT: oosit dış zarf kültür kabı sıkıca kalan ve kolayca oosit ayrılabilir. Bazı oosit kendiliğinden dış zarfları kaybedecek ve soyulmuş oosit olarak telafi edilecektir. Bu prosedür oosit küresel görünümünü korur nispeten istikrarlı bir hazırlık olur. </li></ol> Oosit etrafında 4. Hızlı Çözüm Değişikliği <ol><li> 9,26 tarif edildiği gibi, bir voltaj kelepçesi deneyi gerçekleştirmek. </li><li> deney gereği perfüzyon anahtarları çevirerek yerçekimi beslenen perfüzyon sisteminin çözümünü değiştirin. <ol><li> oositin etrafında agonist konsantrasyonunu hızlı değişiklikler izin vermek için, oosit yüzeyinden yaklaşık 0.4 mm yerleştirilir ağzı olan 1.35 mm&#39;lik bir iç çapa sahip bir cam kılcal yoluyla 6 mL / dakika perfüzyon çözeltisi uygulanır. NOT: değişim hızı önceden tahmini olmuşturaz 0,5 s 27 70% olarak, d. </li></ol></li></ol>

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The authors have nothing to disclose.

Bu makalede açıklanan yöntemler geleneksel 7-9 kullanılan sapma. 1 ila 2 saat kollajenaz muamelesi 8 yumurtalık lobları maruz standarttır; hasarsız, soyulmuş oosit izole; ve mRNA ticari enjeksiyon cihazlarını kullanarak bunları enjekte edin. Oositler, kollajenaz yüksek konsantrasyonda uzun maruz bırakılma ile zarar olması muhtemeldir 1): Klasik bir prosedür, aşağıdaki dezavantajları vardır. 2) kararsız soyulmuş oosit deney kadar saklanmalıdır. 3) Köreltilmiş oosit kökle...

Xenopus oositleri yaygın (- 3, ve oradaki referanslar Kaynaklar 2) bir ekspresyon sistemi olarak kullanılmaktadır. Onlar düzgün montaj ve plazma zarlarının içine fonksiyonel olarak aktif birden fazla alt proteinleri dahil edebiliyoruz. Bu sistemi kullanarak, fonksiyonel olarak, tek başına ya da başka proteinlerle birlikte zar proteinleri araştırılması mutasyon, kimerik ya da birleştirilmiş proteinlerin özelliklerini incelemek için, ve potansiyel ilaçların incelenmesi için mümkündür. başka heterolog sentezleme sistemleri üzerinden oosit kullanmanın avantajları dev hücre kolay kullanım, yabancı genetik bilgi Banyo perfüzyonu ile oositin bir ortamda kolayca kontrol eksprese eden hücrelerin yüksek oranda ve membran potansiyelinin kontrolü, . Bu ifade sisteminin dezavantajı birçok laboratuvarda 17-20 gözlenen mevsimsel çeşididir. Bu varyasyon nedeninet olmaktan çok uzaktır. Buna ek olarak, oosit kalitesi genellikle güçlü bir değişiklik gözlenmiştir. Geleneksel yöntemler 7-9 yumurtalık loblar izolasyonunu, bazı saat kollajenaz için yumurtalık lobların pozlama, soyulmuş oosit seçimi ve oosit mikroenjeksiyon dahil ettik. Burada, alternatif, hızlı prosedürlerin bir dizi bize oosit kalitesinde mevsimsel değişimi ve küçük farklılıklar ile 30 yılı aşkın için bu ifade sistemi ile çalışmak için izin olduğunu bildirildi. Oosit izolasyonu, mikro enjeksiyon cRNA ile ve foliküler hücre tabakası çıkarılması için anlatılan tadil edilmiş ve geliştirilmiş yöntemler, Xenopus oosit içinde tercih edilen bir proteinin ekspresyonu için kullanılabilir. oositin etrafında orta hızlı çözeltisi değişiklikleri için çok basit bir yöntem, herhangi bir ligand-kapılı iyon kanalı ve taşıyıcıların çalışmaya uygulanabilir.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

Proteinler için bir heterolog sentezleme sistemi olarak Xenopus oosit birinci Gurdon ve diğerleri tarafından tarif edilmiştir. 1 ve yaygın olarak keşfinden beri kullanılan (Referanslar 2-3, ve oradaki referanslar). Yabancı kanal ifadesi için oosit çekici kılan bir özellik endojen iyon kanalları 4 kötü bolluğu. Bu ifade sistemi ligand-kapılı iyon kanalı, aralarında çok sayıda proteinlerin karakterizasyonu için yararlı olduğu kanıtlanmıştır. GABA A Kara kurbağası cinsi oositlerinde reseptörleri ve bunların fonksiyonel karakterizasyonu sentezleme oosit, cRNA, foliküler hücre tabakası çıkarılması ve elektrofizyolojik deneyler hızlı çözüm değişikliklerle microinjections izolasyonu da dahil olmak üzere, burada anlatılmıştır. Işlemler bu laboratuarda 5,6 optimize edilmiş ve rutin 7-9 kullanılan olanlardan sapma bulundu. Geleneksel olarak, soyulmuş oositler hazırlanırRT&#39;de yumurtalık loblar uzun bir kollajenaz muamelesi ve bu soyulmuş oosit ile mRNA ile microinjected edilir. Optimize edilmiş yöntemler kullanılarak, çeşitli membran proteinleri olarak ifade edilmiştir ve rekombinant GABA A reseptörleri 10-12, insan rekombinant klorür kanalları 13, tripanosom potasyum kanallarının 14 ve miyo -inositol taşıyıcı 15, 16 olduğu gibi, bu sistem ile incelenmiştir. Burada ayrıntılı yöntemler, Xenopus oositlerde seçim herhangi bir proteinin ekspresyonu tatbik edilebilir, ve hızlı bir çözüm değişikliğin diğer ligand-kapılı iyon kanallarının incelemek için kullanılabilir.

Xenopus oositleri mekanik bir platin döngü (Şekil 1) kullanılarak ayrılmıştı. 0.5: oosit mRNA 2, δ, 0.5 p, A reseptör alt birimleri 4 a GABA kodlayan microinjected, 2.5 fmol / oosit (Şekil 2). 4 Gün sonra, foliküler hücre tabakaları (Şekil 3) uzaklaştırılmıştır. Oositler voltaj -80 mV&#39;de kenetlendi ve 1 uM 3a, 21-dihidroksi-5α-pregnan-20-on (THDOC), güçlü bir pozitif...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus Oositler: Mikroenjeksiyon için optimize Yöntemler, Foliküler Hücre Katmanlar çıkarılması ve Elektrofizyolojik Deneyler Hızlı Çözüm değişiklikler

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

xenopus-oocytes-optimized-methods-for-microinjection-removal

Research

JoVE Journal

Biology

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K Görüntüleme.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

Video: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

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.

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.

Manipülasyon ve<em&gt; İn Vitro</emVe&gt; Olgunlaşma<em&gt; Xenopus laevis</emIntrasitoplazmik sperm enjeksiyonu izledi&gt; oositler, Embriyonik Geliştirme Çalışması

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

Gelişim Biyolojisi

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.

Konfokal Analizi Kullanımı<em&gt; Xenopus laevis</em&gt; Wnt ve Shh morfogen Gradyanların modülyatorlar Soruşturma

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

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltaj kelepçesi florometrisi<em&gt; Xenopus</emFloresan Doğal Olmayan Amino Asitler Kullanan Oositler

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Biyokimya

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.

Xenopus laevis Oosit, Mikroenjeksiyon

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

Biyoloji

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Görselleştirme RNA Yerelleştirme Xenopus Oosit

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

Fertilization of Xenopus oocytes using the Host Transfer Method

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

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Gübrelenmesi Xenopus oositleri Host Transfer Yöntemi kullanılarak

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

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Fare Oosit Mikroenjeksiyon, olgunlaşması ve ploidi Değerlendirmesi

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

Fonksiyonel Çalışmalar için İnsan Sinaptik Reseptörlerini Yeniden Aktive Etmek için Sinaptik Membranların Mikrotransplantasyonu

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

Nörobilim

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Ksenopus Translation Düşüklüğü Belirlemek için Bir Model Olarak laevis

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Xenopus Oositler: Mikroenjeksiyon için optimize Yöntemler, Foliküler Hücre Katmanlar çıkarılması ve Elektrofizyolojik Deneyler Hızlı Çözüm değişiklikler

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Voltaj kelepçesi florometrisi