Este trabalho foi financiado pelo NIH R01 HG002995 &quot;lavrar o Genoma Zebrafish: uma abordagem genética reversa&quot; a CBM.

Parte A: Soluções de congelamento de esperma 1. Peixe Ginsberg Ringers (Faça novo a cada 3 dias; Ginsburg, 1963) NOTA: ordem de adição é importante para evitar PRECIPITAÇÃO Em 450 ml estéril ddH2O dissolver: <ul><li> NaCl 3,25 g </li><li> KCl 0,125 g </li><li> CaCl2 • 2 H2O 0,175 g Em seguida, acrescentar: </li><li> NaHCO3 0,10 g </li></ul> Trazer um volume final de 500 ml com ddH2O estéril e armazenar a 4 ° C. 2. Médio congelamento (Faça diária fresco) <ol style="list-style-type:lower-roman"><li> SEM Metanol <ul><li> Peixe Ginsburg Ringers 10 ml (temperatura ambiente). </li><li> Leite em pó desnatado 1,5 gm </li></ul></li><li> Com metanol NOTA: ordem de adição é importante para evitar PRECIPITAÇÃO <ul><li> Peixe Ginsburg Ringers 9 ml (temperatura ambiente). </li><li> 1 ml de metanol </li><li> Leite em pó desnatado 1,5 g </li></ul> Depois de montar media congelamento, misture bem por 20 min. em agitador orbital ou rocker. Antes de usar, alíquota em 1 ml tubos eppendorf, evitando bolhas de superfície. </li></ol> Parte B: Materiais necessários para o congelamento de esperma Materiais necessários para o congelamento de esperma <ol><li> 10 ml Pipetas descartáveis ​​(Fisherbrand # cat 22-358697) </li><li> Copos de 250ml de água de peixe contendo MESAB / tricaina </li><li> vidros de relógio (Pyrex cat # 9985-75) </li><li> P20 Pipetman (ou equivalente) e dicas </li><li> Esponja titular de peixe (para segurar enquanto aperta masculino) </li><li> Dissecando microscópio com iluminação de palco acima </li><li> Forceps plana (por exemplo, tipo Millipore) </li><li> 2,0 ml frascos criogênicos (Corning cat # 430488) </li><li> 10X10 cryoboxes (Nalgene gato # 03-337-7AA) </li><li> Isopor recipiente com ~ 15 cm de finamente trituradas ICE1 seca </li><li> 15 ml cônico tubos (Falcon # 352099) </li><li> Dewar frasco contendo nitrogênio líquido </li><li> Pinças longas (por exemplo, CMS Fisher gato Cuidados de Saúde n º 10-316B) </li><li> Cryogloves </li><li> Tanque de recuperação para os homens </li><li> Um tubo eppendorf cheia de mídia congelamento sem metanol </li><li> Um tubo eppendorf preenchido com media de congelamento com metanol </li><li> Cryofreezer (eg Taylor-Warton K-series) </li></ol> 1 Um método simples para fazer finamente trituradas gelo seco é envolvê-la em uma toalha e pulverizá-la com um martelo. Um método mais eficiente é usar um moedor de gelo seco, por exemplo, modelo Clawson RE-2. Parte C: método de congelamento de esperma <ol><li> MARK TUBOS CAPILARES: Antes de começar, marque a tubos capilares 10μl com uma caneta de laboratório ao nível de 3,33 mL (ou seja, 16,7 milímetros de baixo, ver Fig. 1.1.). </li><li> Anestesiar MASCULINO: masculino Place (s) no copo contendo MESAB / tricaina diluído em água de peixe (receita em &quot;O Livro dos Zebrafish&quot;) </li><li> PEIXE SECO: Uma vez anestesiados, levante peixe fora de anestésico usando uma colher de plástico e suavemente blot peixe seco numa toalha de papel, com especial atenção para a secagem do lado ventral. Água ativa de esperma por isso é importante para secar completamente em torno da cloaca. Peixes posição no suporte de esponja, acima do lado ventral (Fig. 1.2). Se região em torno de barbatana anal é ainda úmida, secar delicadamente com uma kimwipe. Tome cuidado para não apertar o esperma prematuramente! </li><li> Lugar marcado tubo capilar em borracha adaptador pipeta boca que está incluído com tubos capilares. O adaptador é uma mangueira de borracha que tem um bocal em uma extremidade e um suporte capilar, de outro. Alternativamente, o tubo capilar pode ser conectado a uma seringa Hamilton 50μl através de um pequeno pedaço de tubo de tygon do diâmetro apropriado. </li><li> Masculino lugar no âmbito e expor a abertura urogenital, cuidadosamente espalhar as barbatanas pélvicas além usando a extremidade do tubo capilar. </li><li> Coletar esperma: esperma por expulsar gentilmente acariciando os lados do peixe com uma pinça suave (por exemplo, Millipore), ou apertando suavemente os lados do peixe entre o dedo indicador eo polegar. Coletar o esperma em tubo capilar, uma vez que é expelido através de sucção suave (Fig. 1.2). Evite fezes que pode ser expulso com o esperma. </li><li> Normalizar o volume esperma para 3,3 mL: Se o volume de esperma atinge ou ultrapassa marca de caneta no tubo capilar (ou seja, 16,7 milímetros ou mais), então vá para a etapa 8. Se o volume do esperma não atingir o volume alvo, então normalizar o volume para marcar caneta usando Médio congelar sem metanol (Fig. 1.3). Quantidade mínima de esperma que é aceitável varia com a qualidade do esperma. Esperma bom é branco e opaco. Escassa de esperma parece aguado. Em geral, aceitamos como mínimos: 1 mL de esperma bom (ou 1 / 3 da meta) e 2 mL de esperma pobres (ou dois terços da meta). Volume agora é normalizada para 3,3 mL. </li><li> ADD crioprotetor ao esperma: Suck up Medium congelação com Metanol para a marca laranja (aprox. volume total é agora 20 l;. Fig 1.4). Expulsar mistura de esperma e crioprotetor em área limpa de um vidro de relógio, com especial atenção para não introduzir bolhas. Misture suavemente pipetando cima e para baixo ~ 4 vezes (evitar a introdução de bolhas). </li><li> LOCAL DE ESPERMA 10μl em cada um dos dois criotubos: Pipetar 10 mL do esperma: solução crioprotetor no fundo de duas criotubos separados que têm sido rotuladas com a informação relevante (Fig. 1.5). Movendo-se rapidamente, frascos de tampa e coloque-os na parte inferior da temperatura ambiente de 15 ml tubos Falcon e um cryovial / tubo. Tampa do tubo Falcon e insira o par de tubos cryovial contendo Falcon em gelo seco esmagado. </li><li> FREEZE ESPERMA DE 20 MIN. Em gelo seco: O gelo seco deve ser em forma de pó fino, não pellets. Os tubos devem ser inseridos no gelo seco profundo o suficiente para que apenas seus limites show (Fig. 1.6). , A fim de manter o controle de tubos em gelo seco, dê a cada par de tubos Falcon um número 1-20 (escrito nas tampas) e registrar o tempo eles vão para o gelo seco. </li><li> Criotubos PLACE em nitrogênio líquido:. Após o esperma foi congelado em gelo seco por 20 min, transferi-los para um contendo nitrogênio líquido frasco de Dewar. O esperma é armazenado aqui até a hora de colocar em freezer de nitrogênio líquido. Ao colocar nas caixas de freezer, caixa de freezer lugar em um banho de nitrogênio líquido para que as amostras não aquecer. Use pinças de metal compridas para recuperar frascos de frasco dewar e lidar com cryogloves. Alternativamente, criotubos podem ser transferidos diretamente de gelo seco na caixa se a caixa de freezer freezer é mantida imersa em nitrogênio líquido em um recipiente de isopor. </li><li> Criotubos armazenar a longo prazo em um freezer criogênico de nitrogênio líquido. Para manter a viabilidade de espermatozóides, é importante que os frascos são armazenados imersos em nitrogênio líquido. Em nossa experiência, os frascos armazenados na fase vapor perdem viabilidade ao longo do tempo. A velocidade é importante! O tempo entre a adição do crioprotetor e colocando frascos de esperma em gelo seco (ou seja, as etapas 6-10) deve ser superior a 30 seg. </li></ol> Parte D: Método Fertilização In Vitro Utilizando Espermatozóides criopreservados Nós achamos que isso funciona melhor com duas pessoas. Uma pessoa aperta os ovos de fêmeas, enquanto que a outra pessoa derrete o esperma. <ol><li> Definir um banho de água a 33 ° C. </li><li> Retire do freezer cryovial nitrogênio líquido e transferência para nitrogênio líquido contendo frasco dewar até estar pronto para descongelar. </li><li> Squeeze ovos de fêmeas em 35 milímetros placa de Petri de plástico. Se possível, tente obter três garras de ovos e combinam em um prato. Se você não pode obter três garras bem dentro de 1 min de sua primeira embreagem, então vá para a etapa 4 (uma embreagem boa é suficiente). Definição de embreagem boa:&gt; 150 ovos uniformemente bom olhar (amarelada ou seja, sem detritos branco indicativo de degradação). Mantenha prato coberto, enquanto o esperma é descongelado. </li><li> Remover o frasco de nitrogênio líquido e retire a tampa. Frasco rapidamente mergulhe ~ 1 / 2 caminho em 33 ° C banho de água por 8-10 seg. </li><li> Adicionar rapidamente 70 mL à temperatura ambiente Hanks para o frasco e misture pipetando para cima e para baixo. Imediatamente adicionar aos ovos e misture delicadamente com a ponta da pipeta. </li><li> Sem demora, ative o esperma e os ovos, adicionando 750 mL de água de peixe. Agite para misturar. Incubar 5 minutos à temperatura ambiente. </li><li> Após 5 min, preencha prato com água de peixe e prato de incubar a 28 ° C. Depois de 2-3 horas, contagem e transferência de embriões fértil para pratos de 100mm (50/dish). Contagem de ovos inférteis, para que a fertilização de freqüência pode ser determinada. </li><li> Cuide bem das larvas! Para os primeiros 5 dias, mudar a água diariamente e eliminar os embriões mortos. Coloque apenas larvas que têm bexigas natatórias em berçário no dia 5. </li></ol> E parte: Resultados Representante Nós usamos esta metodologia crypreservation para fazer uma biblioteca de 8.600 amostras de esperma congelado para lavrar entre os anos de 2001 e 2003. Temos descongelados 106 dessas amostras e determinada a sua fertilidade. Fertilidade é a relação de embriões viáveis ​​produzidos em uma fertilização in vitro no dividido pelo número total de ovos que foram fertilizados com o esperma congelado. Frescos (não cryporeserved) esperma normalmente tem&gt; de fertilidade de 95%; espermatozóides criopreservados tem de fertilidade muito menor: uma média de 29% (n = 106 frascos). A variabilidade de frasco para frasco de fertilidade é grande, variando de menos de 1% para tão alto quanto 62%. Esta variabilidade é representada pelas barras de erro representam grande desvio padrão na Figura. 2. No entanto, em 106 amostras de esperma descongelado até à data temos apenas uma vez experimentado 0% de fertilidade tanto em amostras de esperma e não conseguiu recuperar a mutação Tilling correspondente. Pelo descongelamento frascos de esperma da nossa biblioteca ao longo de vários anos (2001-2008), descobrimos que a fertilidade média de amostras de esperma congelado por este método permaneceestável ao longo do tempo (Fig. 2). A fecundidade média das amostras de esperma congelado descongeladas em 2001 foi de 23,5% (com base em 11 amostras de esperma) e em 2008 foi de 25% (com base em 21 amostras de esperma). Uma série de outros pesquisadores agora estão usando esse protocolo de congelamento de esperma em seus laboratórios. Alguns relataram um bom sucesso com as taxas de fertilidade e amostra-to-sample variabilidade semelhante ao que temos observado. Outros têm supostamente não conseguiu fazer este protocolo de trabalho em suas mãos. Acreditamos que as fontes prováveis ​​de problemas são: <ol><li> A não utilização da criotubos exata ou tubos cônicos que sugerimos no protocolo, levando a uma taxa diferente de refrigeração quando em gelo seco; </li><li> A não utilização de gelo seco em pó, levando a uma taxa de resfriamento sub-ótima; </li><li> Usando peixes machos com baixa fertilidade: você deve conseguir pelo menos 1 ml de esperma de um único macho. Você também pode testar a fertilidade masculina, ao fazer uma fertilização in vitro com esperma fresco, no entanto isso pode ser enganador, porque leva muito pouco esperma fresco para chegar perto de 100% de fertilidade. </li><li> Usando óvulos sub-padrão de qualidade na fertilização in vitro. Usando nada além de embreagens perfeito sempre leva para perto de 0% a fertilidade, mesmo a partir de amostras de esperma que realmente têm boa fertilidade (com base na fertilidade do frasco duplicado). </li></ol><img src="/files/ftp_upload/1395/1395fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática da metodologia de congelamento de esperma. <img src="/files/ftp_upload/1395/1395fig2.jpg" alt="Figura 2" /> Figura 2. Fertilidade de espermatozóides criopreservados permanece estável ao longo do tempo. Fertilidade por cento é o número de embriões viáveis ​​produzidos em uma fertilização in vitro, dividido pelo número total de ovos que foram fertilizados com o esperma congelado. &quot;N&quot; refere-se ao número de frascos de esperma criopreservados cuja fertilidade foi testado no ano correspondente. Barras de erro representam o desvio padrão. Uma vez que cada frasco de esperma de um macho é diferente, frasco-ampola para a variabilidade é observada.

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

Uma série de outros pesquisadores agora estão usando esse protocolo de congelamento de esperma em seus laboratórios. Alguns têm relatado a fertilidade semelhantes ea variabilidade de amostra para amostra mesmo que temos observado. Outros têm supostamente não conseguiu fazer este protocolo de trabalho em suas mãos. Acreditamos que as fontes prováveis ​​de problemas são: <ol><li> A não utilização da criotubos exata ou tubos cônicos que sugerimos no protocolo, levando a uma taxa diferente de refrigeração quando em gelo seco; </li><li> A não utilização de gelo seco em pó, levando a uma taxa de resfriamento sub-ótima; </li><li> Usando peixes machos com baixa fertilidade: você deve conseguir pelo menos 1 ml de esperma de um único macho. Você também pode testar a fertilidade masculina, ao fazer uma fertilização in vitro com esperma fresco, no entanto isso pode ser enganador, porque leva muito pouco esperma fresco para chegar perto de 100% de fertilidade. Maiores, machos de cor mais enérgica normalmente dão espermatozóides de qualidade mais elevada. </li><li> Usando óvulos sub-padrão de qualidade na fertilização in vitro. Usando nada além de embreagens perfeito sempre leva para perto de 0% a fertilidade, mesmo a partir de amostras de esperma que realmente têm boa fertilidade (com base na fertilidade do frasco duplicado). </li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
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a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

Este é um método de criopreservação de esperma zebrafish que é uma adaptação do método de Harvey (Harvey et al., 1982). Temos introduziu duas alterações ao protocolo original que tanto racionalizar o processo e uniformidade da amostra aumenta. Primeiro, vamos normalizar todos os volumes de espermatozóides usando a mídia de congelamento que não contém o crioprotetor. Esperma, segundo criopreservados são armazenados em criotubos, em vez de tubos capilares. As taxas de esperma congelamento e descongelamento (δ ° Tempo / C) são provavelmente as duas variáveis ​​mais críticas para o controle deste procedimento. Por esta razão, não substituem os tubos diferentes das especificadas. Trabalhando em equipes de dois, é possível congelar o esperma de 100 homens por equipe, em ~ 2 hrs. Espermatozóides criopreservados utilizando este protocolo tem uma média de fertilidade de 25% (medida pelo número de embriões viáveis ​​gerada em uma fertilização in vitro, dividido pelo número total de ovos fertilizados) e esta fertilidade por cento é estável durante muitos anos.

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

Este é um high-throughput protocolo de criopreservação de esperma para zebrafish. Espermatozóides criopreservados utilizando este protocolo tem uma média de fertilidade de 25% na fertilização in vitro subseqüentes e é estável durante muitos anos.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

30.8K visualizações.  University of California, Davis. Este é um high-throughput protocolo de criopreservação de esperma para zebrafish. Espermatozóides criopreservados utilizando este protocolo tem uma média de fertilidade de 25% na fertilização in vitro subseqüentes e é estável durante muitos anos.

Vídeo: A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Um método de alto rendimento para estudar a morfologia global organela em S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Biologia

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice Cap: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservação do mouse Sperm and Recovery utilizando o I · Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Um Método de alta capacidade para medição de Taxa de filtração glomerular em ratos conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Produção de Haplóide embriões Zebrafish por<em&gt; In Vitro</em&gt; Fertilização

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Um manual de tela pequena molécula Aproximando de alta capacidade Usando embriões Zebrafish

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...