Nosso trabalho na localização RNA é suportado por uma concessão do NIH (R01GM071049) a KLM.

Parte 1: Transcrição de mRNA fluorescente etiquetado. <ol><li> Linearizar DNA plasmídeo contendo o elemento de localização RNA ou seqüência relevante e ressuspender em 1 mcg / mL com DEPC tratadas com H 2 O. O modelo de DNA deve ter sites de promotor a montante para a transcrição de T7, SP6, ou RNA polimerase T3. </li><li> Adicionar os seguintes reagentes a um tubo de 1,5 ml estéril: <table><tbody><tr><td> a. 10X Tx buffer (ver M &amp; M) </td><td> 2 l </td></tr><tr><td> b. 20X cap / NTP mix (ver M &amp; M) </td><td> 1 ml </td></tr><tr><td> c. 1 mM Alexa Fluor 546-14-UTP (Invitrogen) </td><td> 1 ml </td></tr><tr><td> d. UTP, [α-32 P] (1 μCi / mL) (Perkin Elmer) </td><td> 1 ml </td></tr><tr><td> e. DEPC-H 2 O </td><td> 11 mL </td></tr><tr><td> f. 0,2 M DTT </td><td> 1 ml </td></tr><tr><td> g. RNasin (Promega) </td><td> 1 ml </td></tr><tr><td> h. DNA modelo linear </td><td> 1 ml </td></tr><tr><td> i. RNA polimerase (Promega) </td><td> 1 ml </td></tr></tbody></table></li><li> Misture delicadamente os reagentes e centrifugar brevemente (10 seg. Numa microcentrífuga). </li><li> Incubar por 2-4 horas a 37 ° C, cobrindo o tubo com papel alumínio para evitar fotodegradação de fluoróforo. </li><li> Adicionar 1 ml 1 mg / ml RNase DNase (Promega) para degradar DNA template. </li><li> Incubar 15 minutos a 37 ° C. </li><li> Adicionar 79 mL 20 mM EDTA (pH 8,0) para parar a reação. </li><li> Retire 1 ml para um tubo separado, rotulados como &quot;input&quot; e salve. </li><li> Reação através de um spin-1 ml G50 coluna (ver Materiais e Métodos), que incluirá unincorporated nucleotídeos, excluindo mRNA de corpo inteiro. </li><li> Concentrar o RNA por precipitação de etanol: <ol class="lalpha"><li> Adicionar 250 mL de etanol 100%, 10 ml 7M CH 3 COONH 4, 1 ml transportadora RNA (tRNA levedura, 10 mcg / mL) ou 1 mL de glicogênio (20 mg / ml). </li><li> Misture bem e leve ao freezer até sólida a -80 ° C durante&gt; 30 minutos ou em gelo seco por 15 minutos. </li><li> Giram em velocidade máxima em microcentrífuga por 15 minutos, decantar o sobrenadante. </li><li> Lavar com 150 mL de etanol 75%. </li><li> Rodada por 3 minutos na velocidade máxima em microcentrífuga sobrenadante, decantar. </li><li> Secar o pellet a 37 ° C por 3 minutos, garantindo que todo o etanol tenha evaporado. </li></ol></li><li> Ressuspender em 11 mL DEPC-H 2 O e remover 1 ml para um tubo separado, rotulados como &quot;incorporados&quot;. </li><li> Determinar a incorporação por cento e diluir o RNA a 50 nM (ver Materiais e Métodos de cálculo). </li><li> O RNA devem ser congeladas em 5 alíquotas mL, para uso único para evitar congelamento / descongelamento e pode ser armazenado por várias semanas a -80 ° C. </li></ol> Parte 2: Preparando-se para microinjeção. <ol><li> Preparação RNA: Descongelar uma alíquota de RNA (50 nM), e desnaturar a RNA a 70 ° C por 3-5 minutos. Giro por 10 minutos em microcentrífuga a temperatura ambiente na velocidade máxima para remover quaisquer partículas, e continuar a gelo. </li><li> Preparação de ovócitos: <ol class="lalpha"><li> Remover cirurgicamente ovário de Xenopus laevis fêmeas (Nasco). </li><li> Peças de acabamento de Xenopus laevis ovário em um tubo de 50 ml contendo 25 ml de solução de colagenase (ver Materiais e Métodos). </li><li> Agitar suavemente a 18 ° C por 15 minutos, ou até oócitos são visivelmente separados do ovário. Devido à variação entre lotes, colagenase tempo de tratamento pode variar e devem ser cuidadosamente monitorizados através de inspeção visual para garantir defoliculation. </li><li> Permitir oócitos para se instalarem no tubo, retire da solução e lave os ovócitos com tampão MBSH (ver Materiais e Métodos). </li><li> Repita mais duas vezes para lavar um total de três lavagens. Este protocolo de isolamento de oócitos é semelhante ao detalhado em Cohen et al., 2009. </li><li> Classificar manualmente estágio III / IV oócitos (Dumont, 1972) em tampão MBSH sob um microscópio padrão de dissecação. Oócitos fase III estão completamente opaco, mas branco, enquanto ovócitos estágio IV são ligeiramente maiores e salpicado com pigmento. Oócitos que são ligeiramente transparentes são demasiado jovens e ovócitos que totalmente pigmentadas ou apresentam distribuição do pigmento polarizada são muito antigas. </li></ol></li><li> Preparação da agulha: Puxe e bisel agulhas para um diâmetro externo de ~ 0,05 mm. (Nós usamos Drummond Científico de 3,5 polegadas capilares (ordem # 3-000-203-G / X), um Instrumento de Sutter Co. micropipeta extrator e um WPI Inc. beveller.) </li></ol> Parte 3: microinjeção <ol><li> Calibrar agulha com DEPC O 2-H para entregar 2 nl por injeção. Nós frente a nossa carga usando uma agulha microinjetor gás-driven (ver Materiais e Métodos), e calibrar o tamanho da gota usando um micrômetro. </li><li> Carga RNA em agulha. </li><li> Lugar classificadas oócitos em um prato de injeção contendo tampão MBSH. Nós microinject em um prato com uma camada de espuma de borracha preta. Os oócitos pálida se destacam bem em um backgrou brancond. </li><li> Cuidadosamente cada injetar oócitos com 2 nl de RNA em 50 nM. </li><li> Expulsar RNA, lavar a agulha com DEPC O 2-H, e carregar RNA próxima injectável. </li></ol> Parte 4: Cultura de oócitos <ol><li> Oócitos lugar em um poço de uma placa de 24 estéreis bem (Sigma Aldrich). Nós cultura até mil oócitos por poço. </li><li> Remover e substituir tampão com 400 mL O ocyte ULTURA C M edium (OCM; ver Materiais e Métodos) por poço. Coloque placa de cultura dentro de um recipiente hermético plástico com toalha de papel molhado para manter o ambiente úmido durante a cultura. </li><li> Incubar oócitos a 18 ° C para os pontos de tempo que varia entre 8 e 48 horas. </li></ol> Parte 5: Fixação Desidratação e Armazenamento de Oócitos <ol><li> Resolver quaisquer oócitos mortos e coloque oócitos sobreviventes em frascos de vidro. Forma rotineira,&gt; de sobrevivência de 90%. </li><li> Oócitos lugar em MEMFA fixadora (ver Materiais e Métodos) e rock por 20 minutos. Proteger os oócitos da luz, cobrindo com papel alumínio. </li><li> Lavar oócitos, removendo e substituindo fixador com volume igual de tampão MBSH. Repita este lave uma vez mais, num total de duas lavagens. </li><li> Lavar oócitos em metanol anidro: <ol class="lalpha"><li> Retire metade do volume, substitua com metanol. </li><li> Repita o passo &quot;a&quot; duas vezes. </li><li> Retire toda a solução, substitua com metanol. </li><li> Repita lavar metanol. </li></ol></li><li> Oócitos podem ser armazenadas a -20 ° C até que esteja pronto para a imagem. </li></ol> Parte 6: Imagem de localização RNA por microscopia confocal <ol><li> Antes de imagem, adicionar Médio Murray s Clearing (2:1 álcool benzílico benzoato-benzil) para um fundo de vidro FluoroDish (WPI Inc.) para cobrir a superfície de imagem. </li><li> Transferir cuidadosamente os oócitos do metanol para a FluoroDish, minimizando o volume de metanol transferidos. </li><li> Imagem em um microscópio invertido confocal (Nós temos utilizado com sucesso um LSM510 Zeiss e uma Leica TCS SP2). </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Figura 1" /> Figura 1:.. Visualização de Localização RNA Subcelulares pela microinjeção de Transcrições fluorescente etiquetado A. Após a microinjeção, RNA marcado com Alexa Fluor 546 é inicialmente restrita ao núcleo B. Depois de oito horas de cultura, uma RNA fluorescente etiquetado controle pode ser visto de maneira uniforme distribuídos no citoplasma do ovócito. C. No entanto, RNA fluorescente etiquetado contendo seqüências que recrutam as máquinas de transporte pode ser visto no processo de localização subcelular ao pólo vegetal do oócito (para baixo). Barra de escala = 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>

Experimentos com animais de laboratório foram realizados em conformidade com as diretrizes e regulamentos estabelecidos pela Brown University.

Aqui nós apresentamos um protocolo para a visualização de localização mRNA em oócitos Xenopus. Este método, usando transcrições fluorescente etiquetado RNA tem maior relação sinal-ruído do que o anteriormente obtido com marcada com digoxigenina transcrições e é mais simples e mais rápido do que in-situ abordagens baseadas (Mowry e Melton, 1992, Gautreau et al., 1997). Usando este método, podemos engenheiro RNA mutações sequência e rapidamente teste para função in vivo. Al...

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

Erratum: Visualizing RNA Localization in Xenopus Oocytes

Tx tampão 10X <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 espermidina-HCl </li></ul> 20x mix cap / NTP <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 Analógico (New England Biolabs) </li></ul> G-50 coluna <ul><li class="none"> Hidrato de 5 g Sephadex G-50 contas (Sigma Aldrich) em 100 ml deionizada H 2 O. DEPC de tratamento por 30 min. e autoclave. Loja de ações incompletas em temperatura ambiente. Antes de usar, adicione o seguinte RNase soluções: </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 SDS 20% </li><li class="none"> Loja completa G-50 solução a 4 ° C. </li><li class="none"> Remover e descartar o êmbolo de uma seringa de 3 ml (BD Biosciences) e colocar o cano da seringa para um tubo cônico de 15 ml (Corning). Ligue a seringa com uma pequena quantidade de lã de vidro (uma ficha cerca de metade do tamanho de uma moeda de um centavo). </li><li class="none"> Redemoinho solução G-50 completa para voltar a suspender contas. </li><li class="none"> Adicionar 2 ml G-50 solução para a coluna vazia. </li><li class="none"> Rodada por 1 minuto a 1000 x g em centrífuga de bancada. </li><li class="none"> Adicionar 200 mL DEPC tratados com H 2 O deionizada para cada coluna. Spin. </li><li class="none"> Repita mais duas vezes para lavar um total de três lavagens. </li><li class="none"> Remover o corpo da seringa para um novo tubo cônico de 15 ml. </li></ul> Solução de colagenase <ul><li class="none"> 75 mg de colagenase Clostridium histolyticum (Sigma Aldrich) </li><li class="none"> 25 ml 0,1 M KPO 3 + (pH 7,4) </li></ul> MBSH tampão <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 7H 2 O X </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> Meio de Cultura de oócitos <ul><li class="none"> 50% L15 médio </li><li class="none"> 15 mM HEPES (pH 7,6) </li><li class="none"> Uma insulina mg / ml </li><li class="none"> 100 mg / ml de gentamicina </li><li class="none"> 50 nistatina U / ml </li><li class="none"> 50 U / ml de penicilina </li><li class="none"> 50 mg de estreptomicina / ml </li></ul> MEMFA solução <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"> Formaldeído 3,7% </li></ul> Computação rendimento RNA <ul><li class="none"> Determine CPM em &quot;input&quot; e &quot;incorporados&quot; amostras usando um contador de cintilação padrão. </li><li class="none"> incorporação = (&quot;incorporada&quot;) / (10 x &quot;input&quot;) </li><li class="none"> Valores incorporação típicos variam entre 0,03 e 0,10 ~. </li><li class="none"> Rendimentos máximos teóricos para polimerases diferentes: T7, T3, SP6 - 2,64 mg </li><li class="none"> Rendimento da reação em mg = (rendimento máximo de polimerase usada) X (incorporação) </li><li class="none"> Diluir RNA a 50 nM = (mg RNA) / 320 / (comprimento de RNA em bases) / (5X10 -8) </li><li class="none"> A reação geralmente produz ~ 5-10 mL de RNA em 50 nM. </li></ul>

visualizing rna localization in xenopus oocytes

Localização de RNA é um mecanismo conservado do estabelecimento de polaridade celular. Vg1 mRNA localiza-se o pólo vegetal de oócitos Xenopus laevis e age para restringir espacialmente expressão gênica de Vg1 proteína. Um controlo apertado da Vg1 distribuição desta forma é necessário para especificação apropriada germe camada no embrião em desenvolvimento. Elementos de seqüência RNA na 3 &#39;UTR do mRNA, o elemento de localização Vg1 (VLE) são necessários e suficientes para transporte directo. Para estudar o reconhecimento e transporte de Vg1 mRNA in vivo, temos desenvolvido uma técnica de imagem que permite a análise extensiva de trans-fator de mecanismos de transporte através de uma leitura dirigida visual simples. Para visualizar a localização RNA, podemos sintetizar RNA fluorescente etiquetado VLE e microinject esta transcrição em oócitos individual. Após o cultivo de oócitos para permitir o transporte do RNA injetado, ovócitos são fixos e desidratado antes de imagens por microscopia confocal. Visualização dos padrões de localização mRNA fornece uma leitura para o monitoramento da via completa de RNA de transporte e para a identificação de papéis na direção de RNA de transporte para cis-acting elementos dentro da transcrição e trans-acting fatores que se ligam ao VLE (Lewis et al., 2008, Messitt et al., 2008). Nós estendemos esta técnica através de co-localização com RNAs adicionais e proteínas (Gagnon e Mowry, 2009, Messitt et al., 2008), e em combinação com a perturbação de proteínas motoras eo citoesqueleto (Messitt et al., 2008) para investigar mecanismos subjacentes localização mRNA.

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

Visualizing RNA Localization in Xenopus Oocytes

Visualização de<em&gt; In vivo</em&gt; RNA de transporte é realizado por microinjeção de transcrições fluorescente etiquetado RNA em<em&gt; Xenopus</em&gt; Oócitos, seguido por microscopia confocal.

Visualizando Localização RNA em Xenopus Oócitos

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 visualizações.  Brown University. Visualização de In vivo RNA de transporte é realizado por microinjeção de transcrições fluorescente etiquetado RNA em Xenopus Oócitos, seguido por microscopia confocal.

Vídeo: 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.

Gravação de patch-clamp de canais iônicos Expresso em Oócitos Xenopus

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

Biologia

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.

Microinjeção de Oócitos Xenopus laevis

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.

Fertilização de Oócitos Xenopus Utilizando o método de transferência do host

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.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

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.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

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.

Medindo o transporte de catiões de Na, K e H, K-ATPase em<em&gt; Xenopus</em&gt; Oócitos por espectrofotometria de absorção atômica: uma alternativa para Ensaios de Radioisótopos

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.

Transplantes faciais em<em&gt; Xenopus laevis</em&gt; Os embriões

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 Oócitos: Métodos otimizados para microinjeção, remoção de camadas de células foliculares e rápidas mudanças Solução em experimentos eletrofisiológicos

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: Epigenoma célula tipo-específicas de criação de perfil

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.