Name: xenopus laevis as a model to identify translation impairment
Uploaded: 2015-09-27T14:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

Todos os experimentos foram realizados de Xenopus no biotério da Universidade de Lille 1 de acordo com as regras estabelecidas nas orientações do Conselho da Comunidade Europeia (86/609 / CEE) para a experimentação em animais de laboratório. O protocolo animal foi aprovado pelo comitê de ética local (Comité d&#39;en Ethique Experimentação Animale Nord-Pas-De-Calais, CEEA 07/2010). 1. Manipulação de Oócitos <ol><li> Preparar a solução anestésica: dissolver 1 g de m...

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C., Jia, H. 2., Stankowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+protein+s15+phosphorylation+mediates+LRRK2+neurodegeneration+in+Parkinson's+disease.">Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease.</a> Cell. 57 (2), 472-485 (2014).</li><li>Cohen, S., Au, S., Pant&#233;, 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> J Vis Exp. 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A tradução é um mecanismo envolvido na fisiopatologia de numerosas doenças humanas, incluindo várias doenças neurodegenerativas. Por exemplo, na doença de Parkinson vários relatórios sugeriram o comprometimento em tradução associada a mutações hereditárias 8,12,13. Vários modelos celulares estão disponíveis para estudar a tradução. Aqui, a fim de estudar as consequências de translação de uma mutação em eIF4G1 que actua como dominante negativo mutação redu...

As proteínas são elementos essenciais da vida celular e, portanto, em maior escala do organismo. Eles garantem a maioria das funções celulares, incluindo a estrutura, o transporte, a catálise da reacção, a regulação, a expressão do gene, etc. A expressão é o resultado de um complexo mecanismo de tradução permitindo a conversão de um ARNm em proteína. A tradução é submetido a vários controlos para adaptar e para regular a expressão de genes de acordo com as necessidades das células, durante o desenvolvimento e a diferenciação, envelhecimento, stress fisiológico ou manifestações patológicas. A tradução é dividido em 3 fases (iniciação, alongamento e terminação) e apresenta 3 sistemas de tradução de iniciação, a fim de responder a essas necessidades: dependente da tampa, independente-cap via Segmento Ribossomo Entrada interno (IRES) Estruturas e realçadores de tradução cap-Independent ( CITE). A maioria dos mRNA eucarióticas são traduzidos em um tampão-Dependent maneira através da tampa de 5&#39;-trifosfato de 7-metilguanosina, que serve como uma função de reconhecimento, durante a síntese de proteínas. Este tampão liga-se a eIF4E, um componente do complexo eIF4F com eIF4G1 e eIF4A. Associado com outros parceiros como poli (A) Binding Protein (PABP), eIF2 GTP-Met-Met-ARNt, estes factores de iniciação da tradução do mRNA permitir a circularizar e melhorar a sua acessibilidade para as formas 43S complexo de iniciação até que o codão AUG reconhecimento 1. Este caso corresponde ao fim da iniciação da tradução ou seja, o primeiro passo de tradução. Tradução independente de Cap é usado por codificação de mRNA para as proteínas essenciais em condições de tensão que induzem para a proliferação celular e apoptose instância. Este mecanismo envolve estruturas secundárias no ARNm 5&#39;-região não traduzida (UTR) chamado IRES, a extremidade carboxi-terminal de eIF4G1 associado com o complexo eIF4A e 43S. A ligação deste 43S pré-iniciação complex para IRES inicia a tradução tampão independente, sem a necessidade de um factor de 2,3 eIF4E. Por fim, outro mecanismo de tradução ainda não é bem compreendida suporta esta atividade tradução independente-tampão em condições de tensão via CITE estruturas localizadas dentro de mRNA UTR 4. Através destes diferentes modos de tradução que diferem em suas etapas de iniciação, tradução desempenha um papel crítico na homeostase celular e alterações em qualquer um destes processos, assim, teria impacto do organismo com pequena para efeitos de grande escala. Na verdade, o início é um passo limitante da taxa que rege os processos de tradução corretas de mRNA em proteínas e é, portanto, alvo de inúmeros controles e pontos de regulação 5. Se é para o último ou para os componentes desses processos, se acaba por ser defeituoso, ele vai perturbar o equilíbrio estabelecido na célula e, portanto, poderia levar a condi patológicações. Neste contexto, as mutações em fatores de conversão ter sido envolvido em várias doenças, incluindo doenças neurodegenerativas, tais como `Leucoencefalopatia com desaparecer matter&#39; branco (eIF2B1-5 subunidade) 6, na síndrome de Walcott-Rallison (gene que codifica para EIF2AK3 PERK) 7, potencialmente doença (eIF4G1 p.R1205H) de Parkinson 8. Por conseguinte, é importante a realização de estudos celulares e moleculares destas proteínas mutantes para aumentar o nosso conhecimento sobre o desenvolvimento da doença e em geral o processo de iniciação da tradução. Para realizar esses estudos, é essencial para escolher os modelos mais adequados para observar as consequências dessas mutações oócitos de Xenopus laevis são particularmente bem adaptado devido às suas propriedades fisiológicas e bioquímicas:. Sincronicidade fisiológica (bloqueado na fase G2 do ciclo celular) , elevada capacidade de síntese de proteína (200-400 ng / dia / oócito), elevado número de OOC extraídaytes a partir de um mesmo animal (800-1.000 oócitos / feminino) e o tamanho das células (1,2-1,4 mm de diâmetro), o que facilita a sua manipulação. A microinjecção de oócitos de Xenopus com ARNm sintetizado pode ser facilmente realizada para dissecar passos de tradução. Neste ponto de vista, apresenta outras vantagens. Dada a velocidade de progressão da meiose e da tradução do mRNA após microinjecção (~ 24 h), oócito de Xenopus representa um sistema rápido em comparação com os sistemas celulares reconstituídos (extraído de E. coli, germes de trigo ou de reticulócitos de coelho ...) em que um ARNm é traduzida com uma taxa de tradução reduzida e a uma velocidade mais baixa. Assim, os efeitos de uma mutação introduzida em um ARNm irá ser rapidamente e facilmente observável estudada em vários ovócitos. Outra vantagem de oócitos de Xenopus é que mRNAs maternas são latentes e tradução da proteína é bloqueada antes da estimulação progesterona. A adição de progesterona é, portanto, um bom meio de controlar a indução de tradução. Citoplasmática pO olyadenylation não ocorrer durante a oogenese. Inicia-se durante a maturação de oócitos em oócitos de progesterona estimulada por em uma ordem temporal e continua durante todo o desenvolvimento precoce e pode ser usado para estudar as diferentes etapas de tradução. A poliadenilação do mRNA é mos entre os primeiros a ocorrer e que pertence Aurora com A / EG2, Histone-Like B4 mRNA para a classe de genes &quot;de maturação precoce&quot;, definidas na Charlesworth et al. (2004) 9. A indução da tradução do mRNA &quot;final&quot;, como ciclina A1 e B1 Cyclin ocorre em torno do tempo de rompimento da vesícula germinativa (GVBD). Mos ARNm codifica uma cinase serina / treonina-proteína. Sua tradução é crucial, uma vez que induz a quinase MAP cascata que indiretamente ativa a maturação do oócito. Com efeito, em resposta à progesterona, poliadenilação do ARNm MOS é reforçada através de um processo que envolve Aurora A / EG2 proteínas reguladoras e outras proteínas de ligação de ARN com the 3? UTR do mRNA de MOS. Este aumento de poliadenilação do ARNm MOS leva a um aumento do nível de proteína MOS, que por sua vez activa MEK1. Este processo medeia a activação da sinalização extracelular regulada-quinase 2 (ERK2) (Figura 1). Esta cascata de sinalização pode então provocar o M-fase de maturação promover factores, um complexo formado pela ciclina B e cdc2-quinase, e, eventualmente, resulta no recomeço da meiose. Portanto, em oócitos de Xenopus laevis, o estudo de ARNm maternais como MOS pode ser facilmente utilizado para testar a sua possibilidade de tradução com vários parâmetros da sua poliadenilação eficiente para a tradução de vários componentes MOS de sinalização, incluindo também a determinação da taxa de GVBD. Este sistema é, portanto, interessante para avaliar as primeiras conseqüências de mutações nos fatores de iniciação da tradução, sem interferência do mRNA recentemente transcrito ou de eficiência de transfecção, os problemas que ocorrem frequentemente com eukaryestudos com células óticas. Aqui, um protocolo é estabelecido onde mRNAs eIF4G1 mutantes são microinjeção em oócitos de Xenopus laevis ea tradução do mRNA materno é testada. Na presença de um defeito na progressão GVBD, mos de poliadenilação do ARNm que é essencial para a progressão através do ciclo celular meiótica dos oócitos e para o subsequente tradução de mRNAs precoces e tardios classe é verificada. A fosforilação Aurora A / EG2 e ERK também é estudada para confirmar a consequência da mos deregulation.Thus, oócitos de Xenopus representam uma forma simples de analisar diferentes etapas de tradução do mRNA.

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	<tbody>
		<tr>
			<td>Tricaine methane sulphonate</td>
			<td>Sandoz</td>
			<td>MS-222</td>
			<td rowspan="8">oocytes handling</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Moria</td>
			<td>Dumont MC40</td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>Sigma</td>
			<td>781</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T7660</td>
		</tr>
		<tr>
			<td>Veterinarian absorbable Vicryl thread</td>
			<td>Johnson&#38;Johson Intl</td>
			<td>JV1205</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche diagnostics</td>
			<td>10103586001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560S</td>
			<td rowspan="14">Preparation of synthetic mRNA</td>
		</tr>
		<tr>
			<td>DNAse/RNAse free H2O</td>
			<td>Life Technologies</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Merck</td>
			<td>6268</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma</td>
			<td>02854</td>
		</tr>
		<tr>
			<td>Nano Drop</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE 10X</td>
			<td>Eppendorf</td>
			<td>0032006.507</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10 mg/mL</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE&#174; Kit</td>
			<td>Ambion by Life Technologies</td>
			<td>AM1344</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fluka</td>
			<td>03609</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Merck</td>
			<td>1.04003.1000</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
		</tr>
		<tr>
			<td>Gel Doc Imager</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Pipet with 100 8&#34; Glass Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-510-X</td>
			<td rowspan="4">microinjection</td>
		</tr>
		<tr>
			<td>Replacement Glass 8&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-210-G8</td>
		</tr>
		<tr>
			<td>0,45 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLHA025NB</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td rowspan="32">Western Blot</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Biorad</td>
			<td>161-0301</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>A3294</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4612</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L8511</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
		</tr>
		<tr>
			<td>benzamidine</td>
			<td>Sigma</td>
			<td>B6506</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr>
			<td>sodium vanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td>Life Technologies</td>
			<td>NP0323BOX</td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis, mini-Protean TGX</td>
			<td>Biorad</td>
			<td>456-1036 and -1096</td>
		</tr>
		<tr>
			<td>horizontal semi-dry blotting system</td>
			<td>W.E.P. Compagny</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Biorad</td>
			<td>161-0719</td>
		</tr>
		<tr>
			<td>Hybond ECL Membrane</td>
			<td>Amersham Life Science</td>
			<td>10401180</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20846-292</td>
		</tr>
		<tr>
			<td>Ponceau Red (0,5%)</td>
			<td>Sigma</td>
			<td>P3504</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Life Technologies</td>
			<td>A11122</td>
		</tr>
		<tr>
			<td>anti-V5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-58052</td>
		</tr>
		<tr>
			<td>anti-Eg2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-27884</td>
		</tr>
		<tr>
			<td>anti-Eg2-P</td>
			<td>Cell Signaling</td>
			<td>C39D8</td>
		</tr>
		<tr>
			<td>anti-ERK2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1647</td>
		</tr>
		<tr>
			<td>anti-ERK2-P (Tyr204)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7976</td>
		</tr>
		<tr>
			<td>anti-Rsk</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-231</td>
		</tr>
		<tr>
			<td>anti-mos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86</td>
		</tr>
		<tr>
			<td>anti-mouse horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
		</tr>
		<tr>
			<td>anti-rabbit horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2812</td>
		</tr>
		<tr>
			<td>anti-goat horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2378</td>
		</tr>
		<tr>
			<td>Advanced ECL Detection System</td>
			<td>Amershan Life Science</td>
			<td>RPN2135</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td rowspan="11">polyadenylation assay</td>
		</tr>
		<tr>
			<td>TRIZOL (Qiazol Lysis Reagent)</td>
			<td>Qiagen</td>
			<td>79306</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>31998-8</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>278475-1L</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr>
			<td>RTL buffer&#160;</td>
			<td>Qiagen</td>
			<td>79216</td>
		</tr>
		<tr>
			<td>RNAse free DNAse set</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1&#160;</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
		</tr>
		<tr>
			<td>High capacity c-DNA Reverse Transcription kit</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4368813</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Life Technologies</td>
			<td>10342020</td>
		</tr>
	</tbody>
</table>

xenopus laevis as a model to identify translation impairment

A síntese de proteínas é um processo fundamental para a expressão do gene impactando processos biológicos diversos, nomeadamente de adaptação a condições ambientais. A etapa de iniciação, que envolve a montagem das subunidades ribossomais no mRNA codão de iniciação, fator de iniciação envolvidos, incluindo eIF4G1. Defeitos neste passo limitante da taxa de tradução estão ligados a diversos distúrbios. Para estudar as consequências potenciais de tais desregulamentações, oócitos de Xenopus laevis constituem um modelo atraente com um alto grau de conservação dos mecanismos celulares e moleculares essenciais com humano. Além disso, durante a maturação meiótica, os oócitos são transcriptionally reprimida e todas as proteínas necessárias são traduzidos do pré-existentes, mRNAs maternos. Este modelo de baixo custo permite mRNA exógena para tornar-se perfeitamente integrado com uma tradução eficaz. Aqui é descrito um protocolo para avaliação de tradução com um fator de interesse (aqui eIF4G1) usando stored ARNm materno que são as primeiras a ser traduzido poliadenilado e durante a maturação do oócito como uma leitura fisiológico. Em primeiro lugar, o ARNm sintetizado por transcrição in vitro de plasmídeos de interesse (aqui eIF4G1) são injectados em oócitos e cinética de maturação dos oócitos por detecção de ruptura da vesícula germinal está determinada. O alvo de ARNm materno estudado é a serina / treonina-proteína MOS-quinase. Sua poliadenilação e sua subsequente tradução são investigados juntamente com a expressão e fosforilação de proteínas das mos cascata de sinalização envolvidos na maturação do oócito. Variações do protocolo atual de apresentar defeitos translacionais também são propostos para enfatizar a sua aplicabilidade geral. À luz da evidência de que a síntese de proteína aberrante pode estar envolvido na patogénese de desordens neurológicas emergente, um tal modelo proporciona a oportunidade de avaliar facilmente essa deficiência e identificar novos alvos.

Kinetic maturação de ovócitos de Xenopus e determinação do GVBD percentual de oócitos após 24 horas de estimulação PG (Figuras 2B, 2C): A fim de estudar as consequências de translação da mutação eIF4G1-DN, a resposta ao PG em oócitos de Xenopus laevis microinjectados com ARNc eIF4G1-DN é comparado com eIF4G1-WT e de outras condições de controlo (H2O, GFP). Os controlos permitem avaliar a incidência de microinjecção de oócito...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

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.

Xenopus laevis como um modelo para identificar Tradução Impairment

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

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

JoVE Journal

Biology

Xenopus laevis as a Model to Identify Translation Impairment

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Dissecção do Organizador e Explantes Pólo animal a partir de embriões Xenopus laevis e Montagem de um ensaio de Adesão Celular

Biologia

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Incorporação de plástico e secção de embriões Xenopus laevis

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Obtenção de ovos de Xenopus laevis fêmeas

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparação e fracionamento de Xenopus laevis Egg Extractos

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

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 ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Determinação tecido Usando o Transplante Cap Animal (ACT) em Ensaio Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Produção de<em&gt; Xenopus tropicalis</em&gt; Egg Extrai identificar RNAs associada aos microtúbulos

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Usando plusTipTracker software para medir Microtubule Dynamics em<em&gt; Xenopus laevis</em&gt; Cones de Crescimento

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...