RNA lokalizasyonuna KLM NIH (R01GM071049) bir hibe ile desteklenmektedir.

Bölüm 1: floresan ile işaretlenmiş mRNA Transkripsiyon. <ol><li> Plazmid DNA, RNA yerelleştirme elemanı ya da ilgili diğer dizisi içeren Linearize ve 1 mcg / ml DEPC tedavi H 2 O. ile tekrar süspansiyon DNA şablonu T7, SP6 veya T3 RNA polimeraz tarafından transkripsiyon için upstream organizatörü sitelerine sahip olması gerekir. </li><li> 1.5mL steril bir tüpe aşağıdaki reaktifler ekleyin: <table><tbody><tr><td> a. 10X Tx tampon (M &amp; M) </td><td> 2 ul </td></tr><tr><td> b. 20X kapak / NTP mix (M &amp; M) </td><td> 1 ul </td></tr><tr><td> c. 1 mM Alexa Fluor 546-14-UTP (Invitrogen) </td><td> 1 ul </td></tr><tr><td> d. UTP, [α-32 P] (1 μCi / ml) (Perkin Elmer) </td><td> 1 ul </td></tr><tr><td> e. DEPC-H 2 O </td><td> 11 ul </td></tr><tr><td> f. 0.2 M DTT </td><td> 1 ul </td></tr><tr><td> g. RNasin (Promega) </td><td> 1 ul </td></tr><tr><td> h. doğrusal DNA </td><td> 1 ul </td></tr><tr><td> i. RNA Polimeraz (Promega) </td><td> 1 ul </td></tr></tbody></table></li><li> Reaktifler hafifçe karıştırın ve kısaca santrifüj (10 sn. Mikrosantrifüj). </li><li> Tüp fluorofor, photobleaching önlemek için alüminyum folyo ile kaplama, 37 ° C&#39;de 2-4 saat inkübe. </li><li> DNA aşağılamak için 1 ul 1 mg / ml RNaz ücretsiz DNaz (Promega) ekleyin. </li><li> 37 az 15 dakika inkübe ° C </li><li> Reaksiyonu durdurmak için 79 ul 20 mM EDTA (pH 8.0) ekleyin. </li><li> &quot;Giriş&quot; ve kaydetmek olarak etiketlenmiş ayrı bir tüp, 1 ul çıkarın. </li><li> 1 ml G50 sütun üzerinden tam uzunlukta mRNA hariç adi nükleotid içerecektir Spin reaksiyonu (Gereç ve Yöntem). </li><li> RNA etanol yağış Konsantre: <ol class="lalpha"><li> 250 ul% 100 etanol, 10 ul 7M CH 3 COONH 4, 1 ul taşıyıcı RNA (maya tRNA, 10 mcg / ml) veya 1 ul glikojen (20 mg / ml) ekleyin. </li><li> İyice karıştırın ve katı kadar donma -80 ° C&gt; 30 dakika veya 15 dakika boyunca kuru buz üzerinde. </li><li> 15 dakika, süzün süpernatant mikrosantrifüj maksimum hızda dönerler. </li><li> 150 ul% 75 etanol ile yıkayın. </li><li> Mikrosantrifüj, süzün süpernatant maksimum hızda 3 dakika Spin. </li><li> Tüm etanol buharlaştı sağlamak, 3 dakika boyunca 37 ° ° C pelet kurulayın. </li></ol></li><li> 11 ul DEPC-H 2 O süspanse edin ve &quot;anonim&quot; olarak etiketli ayrı bir tüp, 1 ul kaldırmak. </li><li> Yüzde dahil belirleyin ve 50 nm RNA sulandırmak (Malzeme ve Hesaplama Yöntemleri bakınız). </li><li> Donma / çözülme döngüleri önlemek için tek kullanımlık, RNA 5 ul alikotları dondurulmuş ve -80 ° C&#39;de birkaç hafta saklanabilir. </li></ol> Bölüm 2: Mikroenjeksiyon için hazırlanıyor. <ol><li> RNA Hazırlanışı: RNA (50 nM) bir kısım çözülme ve RNA denatüre 70 ° C, 3-5 dakika. Herhangi bir parçacık kaldırmak ve buz üzerinde tutmak için, oda sıcaklığında mikrosantrifüj maksimum hız 10 dakika boyunca dönerler. </li><li> Oosit Hazırlanışı: <ol class="lalpha"><li> Cerrahi Xenopus laevis kadın (Nasco) yumurtalık çıkarın. </li><li> 25 ml kollajenaz çözüm içeren 50 ml konik bir tüp içine Xenopus laevis yumurtalık Trim parçaları (Gereç ve Yöntem) . </li><li> 18 ° C&#39;de 15 dakika süreyle hafifçe sallayın ya da oosit kadar gözle görülür bir şekilde yumurtalıktan ayrılır. Parti değişimi için toplu iş nedeniyle, kollajenaz tedavi süresi değişebilir ve defoliculation sağlamak için görsel olarak dikkatle izlenmelidir. </li><li> Oosit tüp yerleşmek için izin verin, çözüm kaldırmak ve (Gereç ve Yöntem) MBSH tamponu ile oosit yıkayın. </li><li> Üç yıkama olmak üzere toplam iki kez daha tekrarlayın yıkayın. Bu oosit izolasyon protokolü Cohen ve ark, 2009 yılında bu detaylı benzer. </li><li> MBSH tampon standart bir diseksiyon mikroskobu altında elle tür evre III / IV oosit (Dumont, 1972). Evre IV oosit pigment ile biraz daha büyük ve benekli ise Evre III oosit, tamamen opak ancak beyaz. Biraz şeffaf Oosit tam pigmentli veya sergi polarize pigment dağılımı çok eski olduğunu, çok genç ve oosit. </li></ol></li><li> İğne Hazırlanışı: ~ 0.05 mm dış çapı iğne çekin ve konik. (Drummond Bilimsel 3.5 inç kılcal damarlar (sipariş # 3-000-203-G / X), bir Sutter Instrument Co mikropipet çektirmesi ve TEFE Inc beveller.) </li></ol> Bölüm 3: Mikroenjeksiyon <ol><li> DEPC-H 2 O ile iğne enjeksiyon başına 2 nl sunmak için kalibre edin. Biz önden bir gaz tahrik mikroenjektör (Gereç ve Yöntem) kullanarak bizim iğne yükü, ve bir mikrometre kullanarak damla boyutu kalibre. </li><li> RNA iğne içine yükleyin. </li><li> Oosit sıralaması MBSH tampon içeren bir enjeksiyon çanak içine yerleştirin. Biz siyah köpük kauçuk tabakası ile bir çanak microinject. Soluk oosit beyaz bir backgrou iyi öne çıkıyornd. </li><li> 50 nM RNA 2 nl ile her oosit dikkatlice enjekte edilir. </li><li> RNA DEPC-H 2 O ile durulama, iğne ve enjeksiyon için bir sonraki RNA yüklemek boşaltınız . </li></ol> Bölüm 4: Oosit Kültür <ol><li> Oosit iyi steril bir 24 plaka (Sigma Aldrich) yerleştirin. Biz de başına bin oosit kültürü. </li><li> Tamponu çıkarın ve 400 ul O ocyte C ulture M edium ile değiştirin (OCM, Gereç ve Yöntem) ortalama. Kültür plakasına sırasında nemli bir ortam kültürü korumak için ıslak kağıt havlu ile hava geçirmez bir plastik kap içine yerleştirin. </li><li> 18 ° C, 8 ve 48 saat arasında değişen zaman noktaları için oosit inkübe edin. </li></ol> Bölüm 5: Oosit Fixation, Dehidrasyon ve Depolama <ol><li> Ölü oosit Sıralama ve cam şişe hayatta oosit. Biz rutin olarak&gt;% 90 hayatta kalma gözlemlemek. </li><li> MEMFA fiksatif (Gereç ve Yöntem) ve 20 dakika boyunca kaya yerleştirin oosit. Oosit, alüminyum folyo ile kaplayarak ışıktan koruyun. </li><li> Fiksatif çıkarılması ve MBSH tampon eşit hacmi değiştirilmesi oosit yıkayın. Toplam iki yıkar bir yıkama için bir kez daha tekrarlayın. </li><li> Susuz metanol içine oosit Yıkama: <ol class="lalpha"><li> Hacminin yarısını çıkarın, metanol ile değiştirin. </li><li> &quot;&quot; Iki kez adım tekrarlayın. </li><li> Çözüm çıkarın, metanol ile değiştirin. </li><li> Metanol yıkama tekrarlayın. </li></ol></li><li> Oosit resme hazır kadar -20 ° C&#39;de saklanabilir. </li></ol> Bölüm 6: Konfokal Mikroskopi Görüntüleme RNA Yerelleştirme <ol><li> Görüntüleme önce, görüntüleme yüzeyini kaplayan cam bir alt FluoroDish (TEFE A.Ş.) Murray Takas Orta (02:01 benzil benzoat-benzil alkol) ekleyin. </li><li> Dikkatle transfer metanol hacmi en aza indirerek, metanol FluoroDish oosit transfer. </li><li> Ters bir konfokal mikroskop Görüntü (Biz başarıyla Zeiss LSM510 ve Leica TCS SP2). </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Şekil 1" /> Şekil 1: floresan Etiketli Transkriptler Mikroenjeksiyon Subselüler RNA Yerelleştirme Görselleştirme Alexa Fluor 546 etiketli A. takiben mikroenjeksiyon, RNA ilk çekirdeğini sınırlıdır B. kültür sekiz saat sonra, bir floresan etiketli kontrolü RNA eşit şekilde görüleceği oosit sitoplazmasında dağıtılan C. Ancak, taşıma makine işe dizileri içeren floresan etiketli RNA oosit (dibe doğru) bitkisel direğe hücre içi lokalizasyonu sürecinde olabilir. Ölçek çubuğu = 50 mm.

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

Brown Üniversitesi tarafından belirlenen kurallara ve yönetmeliklere uygun olarak laboratuvar hayvanlarında kullanılarak yapılan deneyler yapıldı.

Burada Xenopus oositleri mRNA yerelleştirme görselleştirmek için bir protokol var. Bu yöntem, floresan etiketli RNA transkript kullanarak önceden digoxigenin etiketli transkript ile elde edilen ve in-situ temelli yaklaşımlar (Mowry Melton, 1992, Gautreau ve ark, 1997) daha basit ve daha hızlı daha yüksek sinyal gürültü oranına sahiptir. Bu yöntemi kullanarak, mühendis dizisi mutasyonlar RNA ve hızlı bir şekilde in vivo fonksiyon testi. Ayrıca, ek Alexa UTP fluorophores kullanarak, ...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X Tx tampon <ul><li class="none"> 60 mM MgCl 2 </li><li class="none"> 400 mM Tris-HCl (pH 7.5) </li><li class="none"> 20 mM spermidin-HCl </li></ul> 20x kapak / NTP karışımı <ul><li class="none"> 10 mM CTP </li><li class="none"> 10 mM ATP </li><li class="none"> 9 mM UTP </li><li class="none"> 2 mM GTP </li><li class="none"> 20 mM G (ppp) G Cap Analog (New England Biolabs) </li></ul> G-50 sütun <ul><li class="none"> Hidrat 5 ml 100 g Sephadex G-50 boncuk (Sigma Aldrich) H 2 O. deiyonize 30 dakika DEPC davranın. ve otoklav. Eksik stok oda sıcaklığında saklayın. Kullanmadan önce, aşağıdaki RNaz ücretsiz çözümleri ekleyin: </li><li class="none"> 0.5 ml 0.2 M EDTA </li><li class="none"> 1 ml 1 M Tris pH: 8.0 </li><li class="none"> 0.5 ml% 20 SDS </li><li class="none"> 4 ° C&#39;de tam G-50 çözüm saklayın </li><li class="none"> Çıkartın ve atın piston 3 ml şırınga (BD Biosciences) ve 15 ml konik tüp (Corning) içine şırınga varil. Cam yünü (bir kuruş yarısı boyutu hakkında bir eklenti) küçük bir miktarı ile şırınga takın. </li><li class="none"> Boncuklar tekrar süspansiyon çalkalanarak tam G-50 çözüm. </li><li class="none"> Boş sütun için 2 ml G-50 çözüm ekleyin. </li><li class="none"> 1 dakika için 1.000 x g benchtop santrifüj Spin. </li><li class="none"> 200 ul DEPC tedavi deiyonize H 2 O, her bir sütun ekleyin. Spin. </li><li class="none"> Üç yıkama olmak üzere toplam iki kez daha tekrarlayın yıkayın. </li><li class="none"> Yeni bir 15 ml konik tüp şırınga varil çıkarın. </li></ul> Kollajenaz çözüm <ul><li class="none"> Clostridium histolyticum (Sigma Aldrich) 75 mg kollajenaz </li><li class="none"> 25 ml 0.1 M KPO 3 + (pH 7.4) </li></ul> MBSH tampon <ul><li class="none"> 88 mM NaCl </li><li class="none"> 1 mM KCl </li><li class="none"> 2.4 mM NaHCO 3 </li><li class="none"> 0.82 mM MgSO 4 X 2 O 7H </li><li class="none"> 0.33 mM Ca (NO 3) 2 X 4H 2 O </li><li class="none"> 0.41 mM CaCl 2 X 6H 2 O </li><li class="none"> 10 mM HEPES (pH 7.6) </li></ul> Oosit Kültür Orta <ul><li class="none"> % 50 L15 orta </li><li class="none"> 15 mM HEPES (pH 7.6) </li><li class="none"> 1 mg / ml insülin </li><li class="none"> 100 mg / ml gentamisin </li><li class="none"> 50 U / ml nistatin </li><li class="none"> 50 U / ml penisilin </li><li class="none"> 50 mg / ml streptomisin </li></ul> MEMFA çözümü <ul><li class="none"> 0.1 M MOPS (pH 7.4) </li><li class="none"> 2 mM EGTA </li><li class="none"> 1 mM MgSO 4 </li><li class="none"> % 3.7 formaldehit </li></ul> Bilgisayar RNA verimi <ul><li class="none"> &quot;Giriş&quot; ve standart bir sintilasyon sayacı kullanarak &quot;anonim&quot; örneklerini BGBM belirleyin. </li><li class="none"> dahil = (&quot;dahil&quot;) / (10 x &quot;giriş&quot;) </li><li class="none"> Tipik kuruluş değerleri ~ 0.03 ve 0.10 arasında değişmektedir. </li><li class="none"> Farklı polimerazları için maksimum teorik verir: T3, T7, SP6 - 2.64 mg </li><li class="none"> Reaksiyon verimi mg = (maksimum verim polimeraz kullanılır) X (dahil) </li><li class="none"> Sulandırınız RNA 50 nM = (mikrogram RNA) / 320 / (üsleri RNA uzunluğu) / (5x10 -8) </li><li class="none"> Reaksiyon genellikle verimi ~ 50 nM RNA 50-100 ul. </li></ul>

visualizing rna localization in xenopus oocytes

RNA lokalizasyonu hücre polarite kurulması korunmuş bir mekanizma. Vg1 mRNA Xenopus laevis oosit ve mekansal Vg1 protein gen ekspresyonu kısıtlamak için eylemler bitkisel direğe lokalize. Bu şekilde dağılımı Vg1 sıkı kontrol gelişmekte olan embriyonun uygun germ tabakası belirtimi için gereklidir. MRNA 3 &#39;UTR RNA dizisi elemanları, Vg1 yerelleştirme elemanı (VLE) gerekli ve doğrudan ulaşım için yeterli. In vivo Vg1 mRNA tanıma ve taşıma çalışma için, basit bir görsel okuma ile trans-faktör yönelik taşıma mekanizmalarının kapsamlı analizini sağlayan bir görüntüleme tekniği geliştirdiler. RNA yerelleştirme görselleştirmek için, floresan etiketli VLE RNA sentez ve bireysel oosit bu transkript microinject. Enjekte edilen RNA taşıma izin oosit kültürü sonra oosit konfokal mikroskobu ile görüntüleme için önce sabit ve kurutulmuş. MRNA yerelleştirme desen Görselleştirme RNA ulaşım tam yol izlenmesi ve cis etkili VLE (Lewis ve ark, 2008 bağlamak transkript ve trans-etkili faktörler içinde unsurları, RNA ulaşım yönlendirmede rolleri tanımlamak için bir okunmasını sağlar Messitt ve ark, 2008). Biz ek RNA ve proteinler (Gagnon ve Mowry, 2009, Messitt ve ark, 2008) ile eş-yerelleştirme yoluyla bu tekniği var ve motor protein ve hücre iskeletinin bozulması ile birlikte prob (Messitt ve ark, 2008) mRNA yerelleştirme altında yatan mekanizmalar.

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

Visualizing RNA Localization in Xenopus Oocytes

Görselleştirme<em&gt; In vivo</em&gt; RNA ulaşım floresan etiketli RNA transkript mikroenjeksiyon yapılır<em&gt; Xenopus</em&gt; Oosit, konfokal mikroskobu ile takip.

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 ...

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

11.5K Görüntüleme.  Brown University. Görselleştirme In vivo RNA ulaşım floresan etiketli RNA transkript mikroenjeksiyon yapılır Xenopus Oosit, konfokal mikroskobu ile takip.

Video: Visualizing RNA Localization in Xenopus Oocytes

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Biyoloji

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 ...

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 ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Na, K ve H, K-ATPaz bölgesindeki tarafından Katyon Taşıma Ölçüm<em&gt; Xenopus</emAtomik Absorpsiyon Spektrofotometresi ile&gt; Oosit: Radyoizotop Tahliller Alternatif Bir

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Yüz Nakli<em&gt; Xenopus laevis</em&gt; Embriyolar

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

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

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 (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Hücre türüne özgü Epigenome profil oluşturma

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.