Gostaríamos de agradecer a Andrea Kremsreiter pela excelente assistência técnica. Trabalho em nosso laboratório é apoiado pelo NIH GM096088 eo Vilas Confiança da Universidade de Wisconsin - Madison.

1. Métodos Yeast luminometria Use BYYT cepa de Saccharomyces cerevisiae. É um yvc1-tok1-derivado de BY4741 (MATA, ura3D0, his3D1, leu2D0, lys2D0, yvc1 :: HIS3, tok1 :: kanMX4). Transformar as células com o plasmídeo que expressa pEVP1/Aeq aequorina Leu-seleccionável e o rato que expressam TRPV4 plasmídeo ura-selecionável p416GPDV4, tal como descrito em 26 Batiza et al. E 24 Loukin et al. Utilizar métodos normalizados de construção dos plasmídeos e transformação 27 . Cepa de levedura e plasmídeos estão disponíveis mediante solicitação. <ol><li> Cultura 2 ml de células de levedura durante a noite a fase pós-logarítmica em um agitador a 30 ° C em Leu-ura-&quot;DCD&quot; meio abandono 28 (uma variante empobrecido-sulfato do meio CMD-leu-ura 29), suplementado com sorbitol 1M . Inocular 200 mL destas culturas em 1,8 ml de meio fresco suplementado com 2 mM de o Luciferin celenterazina. Crescer no escuro à temperatura ambiente por mais de 24 horas sem agitação. </li><li> Medir a osmolaridade da cultura (normalmente a 1.400 mOsM) e outras soluções (a seguir) utilizando um osmómetro de pressão de vapor. </li><li> Aliquota de 20 ul de cultura fresco para um tubo de luminómetro de 12 milímetros para um único tubo do luminómetro. </li><li> Para o choque hipotónico, adicionar 200 mL de uma solução, contendo NaEGTA 25 mM (pH 7,2) ou nomes (pH 7,2) e NaCl 500-100 mM, (osmolaridade total de 1,200-400 mOsM), dependendo do grau desejado de choque. Monitorar continuamente a luminescência antes e pelo menos 120 segundos após o downshock osmótica, interrompido apenas pelo breve operação de diluição. Registrar saída luminometer em um computador desktop como unidades de luminescência relativa (LSD). </li></ol> Embora não seja para o estudo de TRPV4 transgénico, temos usado uma variante do protocolo acima num sistema automatizado para analisar as respostas de um grande número de estirpes de levedura. Estudo of as respostas aos hipo-30 ou 31 hiperosmóticas estímulos do deletome levedura (a coleção de 4.906 levedura deletants de um único gene), utilizando uma luminometer microplaca e analisados ​​com o software correspondente foi bem sucedida. 2. e 3. Ovócitos Métodos Eletrofisiologia Eletrofisiologia usa métodos básicos 32. PCR amplificar a grelha de leitura aberta de tipo selvagem ou mutante utilizando TRPV4 alta-fidelidade PfuUtra polimerase e integrado pGH19 33. Isto implica uma inserção precisa do ORF entre 5 &#39;e 3&#39; UTRs do gene da p-globina de Xenopus β e a jusante de uma polimerase de ARN de T7-promotor. Linearizar o construto a jusante da UTR 3 &#39;e utilizadas como modelos in vitro em T7 ARN polimerase de reacção. Use técnicas de biologia molecular padrão 34. Use estágio V-VI ovócitos de X. laevis. A criação de animais, ovariecto parcialminha, a remoção envelope defolliculation e vitelino siga os procedimentos padrão 32,33,35. Injetar 2-40 ng de solução cRNA por ovócitos utilizando um microinjetor automático Drummond Nanoinject II. Injetar ~ 30 ovócitos para cada conjunto de experimentos. Após injecção TRPV4-ARNc, incubar os oócitos no meio ND96 com gentamicina (100 ug / ml) e 1 pM de ruténio vermelho 14. 2. Dois eletrodos de tensão da braçadeira <ol><li> Puxe borosilicato pipetas de gravação de vidro de precisão descartáveis ​​micropipetas 100 ul individualmente com um puxador de pipeta. </li><li> Puxe os dois eletrodos de pipeta de corrente injeção de tensão de medição e são puxados para ter uma abertura de ponta de ~ 1 mícron de diâmetro. </li><li> Eletrodos de aterramento com 2 M KCl resultando em 0,1-0,2 mohms resistência. Montá-los em headstages HS2A, que em conjunto com um VG-2Ax100 virtual aterrado braçadeira banho, estão ligados a uma interface amplificador GeneClamp 500 através de um digitalizador Digidata 1440. Adquirir<html> dados utilizando software pClamp10. </li><li> Coloque o oócito para ser testado em um banho de 1 ml de uma câmara de plástico fabricada montado na plataforma de um microscópio de dissecação com 20X de ampliação. A solução do banho é virtual fundamentada por uma ponte de ágar 3 M KCl e um fio de prata clorada colocado perto do oócito e ligado a uma fase de terra virtual VG-2A. </li><li> Construa a câmara para a perfusão contínua com tubos de plástico como entrada e saída de solução. Ligar o tubo de entrada está ligado a um sistema de perfusão fabricada, que é um colector de 50 ml de seis reservatórios de seringas, cada um cheio com uma solução diferente. Taxas de alimentação de gravidade de qualquer solução particular é controlada para ~ 2-3 ml / min usando &quot;Keck&quot; grampos rampa nas tubulações. </li><li> Montar os dois eléctrodos com os seus headstages em micromanipuladores. Realize penetrações eléctrodos da ovócito por micromanipulação. </li></ol> 3. Grampo patch Exame de mecanosensibilidade direto &quot;&gt; Os métodos básicos de patch clamp incluindo a preparação pipeta, formação GÊ-selo, e em formação de células ou modos excisadas são aqueles em Hamill et al. 36 e Conn 32. <ol><li> Encha pipetas de vidro de silicato de boro, com uma abertura de diâmetro de ~ 1 mM na ponta (número de bolhas 5-6) com KCl 98 mM, MgCl2 1 mM, e 10 mM de K +-HEPES, pH 7,2. </li><li> Prenda a pipeta patch clamp através de um tubo de plástico para uma seringa de 5 ml e através de uma T-junção, também a um manômetro. Utilize um computador para lidar com ambos o eletrodo e as saídas manômetro. Aquisição de dados em 10 kHz, e, em seguida, reproduzir através de um filtro de oito pólos Bessel em 1 kHz para análise. </li><li> Diferentes remendos podem ser excisadas a partir do mesmo oócito repetidamente. Esta prática faz com que o exame da atividade molecular de TRPV4 mais eficiente e confiável. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Como foi dito na Introdução, os métodos comumente utilizados para estudar funções TRPV4 por vezes resultou em inconsistências e controvérsias. Os dois conjuntos de métodos aqui descritos oferecem algumas vantagens e pode complementar os métodos existentes. Enquanto que apenas descrevem estudos sobre TRPV4, os métodos podem ser estendidos para estudar outros canais iónicos bem. 1. Métodos Yeast luminometria Brotando levedura tem uma Ca 2 +-resposta<...

TRP (potenciais receptores transientes) composto por sete subfamílias de canais de cátions que servem funções sensoriais 1,2. Nos mamíferos, TRPV, subfamília vanilloid de TRP, tem seis variedades. TRPV4 (tipo 4) 3,4 responde ao calor, determinados produtos químicos, inchaço osmótico, ou tensão de cisalhamento. O gene TRPV4 foi repetidamente isolados por candidato a gene e / ou de clonagem de expressão 5-8. O último método seguido resposta do produto de gene a hipo-osmolaridade. TRPV4 é expresso em quase todos os órgãos e funções do desenvolvimento, fisiologia, patologia ou de muitos tipos de células diferentes de 3,4. Striking são os&gt; 50 autossômicas humano mutações TRPV4 dominantes, fazendo com que as neuropatias periféricas e / ou displasias esqueléticas (anormalidades no desenvolvimento do esqueleto) 9-11. As displasias esqueléticas variar de leve tipo brachyomia 3 (tipo 3 nanismo), displasia spondylometaphyseal tipo Kozlowski, a severe displasias, alguns causando morte neonatal ou embrionário. Apesar de todas as formas de mecanismos parecem possíveis, nenhum explica a diversidade, complexidade, variabilidade e sobreposições ocasionais dessas doenças 4. Como outros canais TRP 1, TRPV4 é um tetrâmero. No rato ou TRPV4 humana, cada subunidade é composta de 871 resíduos. O elemento central é o seis transmembranar de hélices (S1-S6), que são susceptíveis arranjados de uma forma semelhante aos canais sensíveis à voltagem de K +. Há, S1 a S4 formar um domínio periférica e a S5 e S6 formar o domínio de permeação de poro. Entre S5 e S6 de TRPV4 é uma hélice de poro curta seguido do LDLFKLTIGMGDL sequência, quatro dos quais presumivelmente convergem para formar o filtro de catião. O 470-resíduo segmento citoplasmático N-terminal contém seis repetições de anquirina, que se sabe ligarem a proteínas ou ligantes. O segmento citoplasmático de 152 resíduos C-terminal inclui uma sequência de ligação à calmodulina, entre outros possíveis locais que se ligam otseus elementos 3. TRPV4 é um canal de cátions que essencialmente exclui ânions 1. Embora a sua função fisiológica é a transdução de estímulos de influxo de Ca2 +, é também permeável a outros catiões com uma sequência de permeabilidade Eisenman IV, favorecendo bivalente numa Ca P: P Na ~ 7 12. Single-channel condutância retifica em ~ 90 pS para fora e para dentro ~ 40 pS 6,13,14. Heterologamente expressa atual (abaixo) pode ser ativado por hipo-osmótico inchaço, tensão de cisalhamento, ou calor 15. Ele também é ativado por ácidos graxos poliinsaturados 16,17 eo éster phorbal sintético 4αPDD 18. No presente, o agonista mais potente é GSK1016790A 19 e antagonista é GSK2193874, eficaz em 10 -9 a 10 -8 M 20, ambos descobertos pela maior taxa de transferência, a tela de pequeno peso molecular. Duas áreas-chave da investigação TRPV4 permanecer confuso: (1) Mesmo quando TRPV4 é amplamente estudada por sua mecanosensibilidade, a sua base molecular é controversa. Um modelo de hipo-osmolaridade de alguma forma activa a fosfolipase A2 (PLA2) para produzir o ácido gordo polinsaturado (PUFA) de ácido araquidônico (AA), que é convertido em ácido epoxyeicosatrienoic (IIT) por uma epoxigenase, e a ligação da EET activa TRPV4 16, 17. No entanto, a própria TRPV4 foi mostrado para responder directamente ao estiramento da membrana 14 (abaixo), fornecendo uma explicação mais simples. (2) As patologias são mutantes TRPV4 desconcertante. Na base, é preciso saber se as doenças são devido à perda, a redução ou o aumento das atividades do canal. Mesmo aqui, a literatura está longe de ser clara. Enquanto alelos múltiplos esquelético-displasia foram relatados para ter atividades mais elevadas, (ganho de função, GOF) 4,21, vários foram relatados para ter reduzido actividades (perda de função, LOF) 10,22. Um estudo sistemático de 14 alelos encontrou-os atodas as mutações ser GOF (abaixo) 23. A alegação de que alguns são LOFs parece contradizer o fenótipo de TRPV4-/ - rato knockout, que são viáveis ​​ou férteis, com apenas pequenos defeitos, apesar de uma perda completa da função TRPV4. Uma parte dessas controvérsias tem origem metodológica. Laboratórios usam diferentes métodos, ou variantes de um método, e empregam diferentes padrões de julgamento. Mais comumente, TRPV4 é transitoriamente expressa em células de mamíferos em cultura (HEK, CHO, HeLa) ea ascensão do interno [Ca 2 +] em cima de agonista ou estimulação hipotônica está registrado com o Ca 2 + corante fluorescente sensível, Fura-2. O excesso de confiança em este ensaio fluorimétrico foi criticado 1. O nível de expressão em diferentes populações, a distribuição no mesmo, bem como a concentração efectiva do corante, necessita de ser controlada e documentada. Mais confiável é os exames eletrofisiológicos diretos. Mesmo isto, como commonly praticado, também não é sem problemas. Porque os níveis de expressão em células de cultura individuais são difíceis de controlar, as correntes de células completas têm grandes variações. Além disso, porque as correntes são pequenos, estatísticas confiáveis ​​terá que contar com grandes tamanhos de amostra, muitas vezes não é prático. Exames de patch-clamp raramente foram executadas. Algumas dessas gravações mostram aglomerados de rajadas de atividade que fazem avaliação estatística desafiador 16,17. Para entender melhor o mecanosensibilidade molecular, nós desenvolvemos dois sistemas adicionais para examinar TRPV4. (1) Para isolar TRPV4 longe de seus parceiros de mamíferos habituais, temos expressa TRPV4 rato em brotamento levedura 24. Expressão funcional de TRPV4 neste contexto evolutivamente distante mostrou que ele ainda pode responder a força osmótica sem os seus parceiros habituais. Porque o fermento não faz PUFAs como AA ou EET, e seu genoma tem genes PLA2 ou epoxigenase, este expression também mostra que não são necessários para TRPV4 para detectar força. Tendo TRPV4 nos moleculares eucariontes biologicamente mais amenas também permite manipulações eficiente para a frente ou reversa genéticos 25. (2) Para as análises em profundidade biofísicos de TRPV4, expressamos TRPV4 em oócitos de Xenopus. Ao contrário das células cultivadas, que produzem sub-nA a nA (10 -10 a 10 -9 A) correntes, um ovócito expressa correntes em mA de (10 -6 a -4 10 A). Sinal muito maior sobre o ruído permite uma melhor quantificação e comparação mais confiante. TRPV4, de modo expresso, também podem ser examinados como moléculas individuais usando uma braçadeira de patch. Um único oócito permite amostragem remendo repetiu, novamente fazendo a quantificação mais confiável. Tais estudos mostraram que o próprio canal TRPV4 podem ser diretamente ativados por força trecho membrana 14. As análises também mostraram que 14 representantes alelos esquelético-displasia são todas as mutações de ganho de função. Além disso, o degree deste Ca 2 + vazamento constitutiva paralela à gravidade das doenças ósseas 23. Por causa de sua novidade e utilidade, os procedimentos detalhados desses dois métodos são montados aqui para permitir repetições, em futuras pesquisas sobre TRPV4 ou canais semelhantes.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4 (Transient Receptor Potenciais, família vanilloid, tipo 4) é amplamente expresso em tecidos de vertebrados e é ativada por vários estímulos, inclusive por forças mecânicas. Certas mutações TRPV4 causar óssea hereditária complexo ou patologias neuronais em humanos. De tipo selvagem ou mutante TRPV4 transgenes são geralmente expressos em células de mamífero de cultura e examinadas por Fura-2 fluorometria e pelos eléctrodos. Em termos de mecanismo de mecanosensibilidade e as bases moleculares das doenças, a literatura atual é confuso e controverso. Para complementar os métodos existentes, descrevemos dois métodos adicionais para examinar as propriedades moleculares de TRPV4. (1) de rato e um transgene TRPV4 aequorina são transformadas em leveduras de brotamento. Um choque hipo-osmtic da população transformante produz um sinal luminométrico devido à combinação de aequorin com Ca 2 +, lançado pelo canal TRPV4. Aqui TRPV4 é isolado a partir dos seus parceiros proteínas de mamíferos normaise revela o seu próprio mecanosensibilidade. (2) ARNc de TRPV4 é injectado em oócitos de Xenopus. Após um período de incubação adequado, a corrente de TRPV4 macroscópica é examinada com uma pinça de voltagem de dois eléctrodos. O aumento de corrente na eventualidade de remoção de osmótico inerte a partir do banho de oócitos é indicativo de mecanosensibilidade. Os microampere (10 -6 a -4 10 A) correntes de ovócitos são muito maiores do que os subnano a nanoampere (10 -10 a 10 -9 A) correntes de células em cultura, produzindo quantificações mais claras e avaliações mais confiantes. Correntes microscópicos que refletem as atividades das proteínas de canais individuais também pode ser registrada diretamente sob um patch clamp, em on-célula modo excisadas ou. O mesmo ovócito fornece várias amostras de patch, permitindo a replicação de melhores dados. Aspiradores aplicados aos patches podem ativar TRPV4 para avaliar diretamente mecanosensibilidade. Esses métodos devem também ser úteis no estudo de outros tipos de canais TRP.

Numa experiência típica luminometria, uma gama de 400-1,200 mOsM hipotónicas choques para baixo é conseguida através da adição de 200 ul de soluções de choque de baixa osmolaridade diferente de 20 l de cultura a 1.400 mOsM. A actividade TRPV4 é evidente quando a RLU do experimental é comparada com a de dois controlos negativos: as células de levedura transformadas com o plasmídeo p416GPDV4 vazio ou um que contenha o gene TRPV4 com uma mutação no filtro de iões (Figura 1) 24...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

Para complementar os métodos habitualmente utilizados para estudar TRPV4 de, dois métodos são descritos: O mecanosensibilidade pode ser estudada por Ca 2 +-aequorina luminometria em leveduras transgénicas após desafio com hipo-osmótico. Ele também pode ser examinada em TRPV4-ARN injectado oócitos de Xenopus a-célula inteira de dois eléctrodos de aperto de voltagem ou remendo grampo em no modo de célula ou excisadas.

Luminométricos levedura e<em&gt; Xenopus</em&gt; ovócitos eletrofisiológicos Exames do mecanosensibilidade Molecular de TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K visualizações.  University of Wisconsin – Madison.  Para complementar os métodos habitualmente utilizados para estudar TRPV4 de, dois métodos são descritos: O mecanosensibilidade pode ser estudada por Ca 2 +-aequorina luminometria em leveduras transgénicas após desafio com hipo-osmótico. Ele também pode ser examinada em TRPV4-ARN injectado oócitos de Xenopus a-célula inteira de dois eléctrodos de aperto de voltagem ou remendo grampo em no modo de célula ou excisadas. 

Vídeo: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Uma técnica rápida para a visualização de células vivas de levedura Imobilizado

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Biologia

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Evolução Molecular do Recombinase Tre

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

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 ...

Visualizing RNA Localization in Xenopus Oocytes

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

Visualizing RNA Localization in Xenopus Oocytes

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

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 ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Produção de transgênicos Xenopus laevis Por Restrição Integração Mediada por Enzima e Transplante Nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Um Ensaio de microscopia de fluorescência para Mitophagy Monitoramento no fermento Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitiva Screens Genomic de Bibliotecas Yeast Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificação cromatográfica de Ribossomos Yeast Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

O Processo de monstro verde para a Geração de linhagens de levedura de transporte deleções de genes múltiplos

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

O<em&gt; Xenopus</em&gt; Oocyte Cut-aberto vaselina Gap Técnica Voltage-clamp Com Fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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