Name: transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform
Uploaded: 2017-01-30T13:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

A parte animal desse estudo foi realizado na Unidade de Investigação Metabólica da Universidade de Alberta, Edmonton, Canadá, com todos os procedimentos experimentais em animais aprovado (Protocolo # AUP00000131) pela Universidade de Alberta Animal Care e do Comitê Use, e os animais tratados de acordo ao Conselho canadense de diretrizes animal Care (1993). 1. Produção de Embriões, Isolamento de RNA total e Tratamento DNase <ol><li> Para protocolos experimentais de animais e de colheita de embriões se referir em nossas publicações anteriores 17, 18. </li><li> Piscina e encaixe congelar embriões estágio similar de cada vaca em nitrogênio líquido e, em seguida, manter a -80 ° C até a extração de RNA. </li><li> Extrair o ARN total por meio de um estojo de extracção de ARN baseada em coluna, que permite extrair o ARN a partir de amostras escala Pico (kit de informação está disponível na lista de materiais). NOTA: Recomendado kit contém: Condicionado Tampão, Extração Buffer, 70% de etanol, tampão de lavagem 1, tampão de lavagem 2, tampão de eluição, colunas de purificação de RNA com tubos de coleta e microtubos. </li><li> Adicionar 10 ul de tampão de extracção contendo embriões de tubo e incubar durante 30 min a 42 ° C. tubo de centrifugação a 800 xg durante 2 min para recolher o extracto de células no tubo de microcentrífuga. </li><li> Pipetar 250 mL de buffer Condicionado para a membrana do filtro de purificação de coluna. Incubar a coluna de purificação de ARN com tampão condicionado durante 5 min à temperatura ambiente. Centrifugar a coluna de purificação no tubo de recolha a 16.000 xg durante 1 min. </li><li> Pipetar 10 ul de 70% de etanol com a mistura de ARN Tampão de Extracção e embriões. Misture bem, pipetando cima e para baixo. mistura pipeta na coluna de purificação pré-condicionados. </li><li> Para ligar ARN, centrifugar a coluna de purificação de 2 min a 100 xg e segue-se imediatamente através de uma centrifugação a 16000 xg durante 30 segundos para remover o fluxo através. </li><li> pipeta 10081; L de tampão de lavagem 1 para a coluna de purificação e centrifugar durante 1 min a 8000 x g. NOTA: DNase tratamento é recomendado se realizar a transcrição reversa ou amplificação após o isolamento do RNA. </li><li> Digerir o ADN genómico direita após o passo 1.8. NOTA: Recomendado kit DNase está disponível na lista de materiais. kit recomendado contém: solução de DNase I e solução tampão. </li><li> Prepare a mistura de incubação com DNase por mistura de 5 mL de ADNase I solução de reserva com 35 mL de buffer. Misture invertendo suavemente. </li><li> Pipetar a pL de DNase mistura de incubação de 40 directamente para a membrana da coluna de purificação. Incubar à temperatura ambiente durante 15 min. </li><li> Pipetar 40 ul de tampão de lavagem 1 para a membrana da coluna de purificação e então centrifugar a 8000 xg durante 15 s. </li><li> Tampão Pipetar 100 mL de lavagem 2 para a coluna de purificação e centrifugar durante 1 min a 8000 x g. </li><li> Pipetar outro 100 uL de tampão de lavagem 2 para a coluna de purificação e centrifugardurante 2 minutos a 16.000 x g. </li><li> Transferir a coluna de purificação para um novo tubo de microcentrifugação de 0,5 mL. Pipetar 11 ul de tampão de eluição directamente para a membrana da coluna de purificação. Para garantir a absorção máxima de tampão de eluição na membrana, tocar suavemente a ponta da pipeta para a superfície da membrana, enquanto a distribuição do tampão de eluição. </li><li> Incubar a coluna durante 1 min à temperatura ambiente. Centrifugar a coluna durante 1 min a 1000 xg para distribuir tampão de eluição na coluna, e em seguida girar durante 1 min a 16.000 xg, para eluir o ARN. </li><li> Use Bioanalyzer instrumento de acordo com as instruções do fabricante para avaliar a qualidade e quantidade de ARN de 19. </li><li> Armazenar a amostra de ARN a -80 ° C até à sua utilização. </li></ol> 2. RNA amplificação e Rotulagem de Análise de Microarray NOTA: Para o primeiro usuário tempo de trabalho com procedimentos de amplificação de RNA e de rotulagem, use cinco ng de pico-in RNA along com as amostras reais arna para controlar a medição da amplificação de ARN e hibridação de controlo de qualidade. O pico-in mix RNA contém dez in vitro sintetizados, transcrições poliadenilados em proporções predeterminadas. Quando o procedimento realizado adequadamente, as transcrições rotulados especificamente hibridizam apenas para sondas de controlo complementares em matrizes e os dados podem ser digitalizados para rastrear garantia de controle de qualidade com a auto-mínimo ou hibridação cruzada. Mais detalhes descrição sobre o procedimento cravado-in intensidades de controle e normalização após a digitalização da análise da matriz e de dados é referir-se a publicações anteriores 20, 21. O procedimento que se segue é dada para os utilizadores de microarranjos de rotina. <ol><li> Prepare (valor de número de ARN Integridade (NIR), pelo menos&gt; 7,0) de 100 pg de alta qualidade amostra de ARN num volume total de 10 - 11 mL. </li><li> Amplificar a amostras de ARN com um estojo de amplificação de ser capaz detrabalhar com amostras escala pico (tão pouco quanto 100 pg). NOTA: Recomendado informações kit de amplificação está disponível em Lista de materiais e segue as instruções do fabricante, sem qualquer modificação. </li><li> Usar uma quantificação à base de fluorescência para avaliar o perfil da gama de tamanhos do ARNA amplificado. </li><li> Utilizar o instrumento de espectrofotômetro (com base na absorvância a 260 e 280 nm) para medir a concentração da ARNA amplificado. </li><li> Armazenar o ARNA amplificado a -80 ° C até estar pronto para rotulagem. </li><li> Use 2 mg de RNA amplificado para marcação fluorescente de acordo com o guia do usuário ULS ARNA kit rotulagem. NOTA: Uma vez que Cyanine 547 e 647 Cyanine corante são sensíveis a foto-degradação e Cyanine 647 corante é facilmente degradada pelo ozono, o procedimento de marcação tem lugar em uma quantidade mínima de luz e sob um ambiente livre de ozono. </li><li> Ligue a caixa de ozono (Figura 1A) até que o nível de ozono é de 0,001 ppm. </li><li&gt; fazer a configuração de reacção dentro da caixa de ozono (Figura 1B) por adição de 2 ul de Cianina 547 ou Cianina 647, 2 uL de tampão de marcação, e, em seguida, ajustar para um volume final de 20 mL com água sem RNase sob a luz de segurança. Adicionar filtro de câmara escura para a fonte de luz. </li><li> Misturar suavemente e incuba-se os tubos em um termociclador a 85 ° C durante 15 min. Colocar as amostras em gelo durante pelo menos 1 min. Girar para baixo para recolher o conteúdo de tubos, antes de prosseguir com o estojo de extracção de ARN, excepto passos relacionados com tratamento de DNase (1,9-1,11), para limpar o ARN amplificado Cy-corante-rotulados. </li><li> Utilizar o instrumento espectrofotómetro para medir e determinar a concentração de ARN amplificado marcado e manter o ARNA marcado à temperatura de -80 ° C no máximo durante 3 dias antes da utilização. </li></ol> 3. Microarray hibridação e de lavagem NOTA: Os seguintes passos importantes são adaptados de acordo com duas cores à base de Microarray Gene Expression Análise de Protocolo (versão6.5, maio de 2010). <ol><li> Adicionar uma quantidade igual de 825 ng de cada Cyanine 547- e Cyanine 647 marcado ARNA e misture com 25x e 10x fragmentação buffers de bloqueio. </li><li> Aquecer a mistura a 60 ° C durante 15 minutos e arrefece-se em gelo durante 1 min. </li><li> Adicionar uma quantidade igual de 2x tampão de hibridização e misturar bem. </li><li> Centrifuga-se a 13000 rpm, em seguida, a mistura está pronta para hibridação. </li><li> Carga de 100 mL da mistura sobre a lâmina de matriz e vedar a corrediça no interior da câmara de hibridação. </li><li> Carregar a câmara montada no forno de hibridação durante 17 h a 65 ° C em rotação a 10 rpm em forno. </li><li> Lave as matrizes de acordo com o protocolo com tratamento de protecção de ozono por imersão em solução de estabilização e secagem durante 30 s. </li><li> Retirar as matrizes a partir da solução e certificar-se da superfície da matriz é seca com as partículas de poeira não há </li></ol> 4. Digitalização Microarray Deslize <ol><li> Mantenha as matrizes no lugar escuro e ligue scannER até que esteja pronta a utilizar (15 min de tempo de espera). </li><li> Coloque o código de barras matriz na esquerda, no scanner e iniciar a digitalização. Fazer a análise local de acordo com o guia do usuário do software e salvar os dados como (GPR) formato de arquivo. </li></ol> 5. Análise Estatística <ol><li> Faça o download do software FlexArray http://genomequebec.mcgill.ca/FlexArray/license.php Clique em &quot;dados brutos de importação&quot; e carregar os arquivos de GPR em FlexArray. </li><li> Realizar a normalização e a análise estatística, se necessário, seguindo as instruções. Nota: o cálculo do limiar para a seleção local positiva neste estudo como foi descrito anteriormente 22. </li><li> Selecione no menu suspenso o manual do visualizador gráfico da FlexArray para ver todos os pontos cruzados e sinais de fundo. Nota: Um quadro semelhante das cravado nos controlos, após hibridação com o micro-arranjo pode ser visto no estudo anterior 8. </li><li> Selecione umanormalização Loess dentro matriz seguinte por um quantil entre o processo de matrizes normalização do FlexArray suspensa manual de conformidade. </li><li> Exportar todos os dados normalizados de FlexArray em um arquivo do Excel para posterior utilização. </li></ol> 6. Análise Bioinformatics A anotação original das sequências de sondas de transcrições específicas de embriões bovinos (BESTv1) microarray foi descrita anteriormente 8. No presente estudo, foram obtidos 20,403 símbolos único gene (GS) ( <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53754/Supplement_file_1.xlsx">Suplemento File1</a> ) através do Sistema de Gestão EmbryoGENE Laboratório de Informação (LIMS) ELMA (http://elma.embryo-gene.ca). Uma lista de 6.765 genes únicos ( <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53754/Supplement_file_2.xlsx">Suplemento File2</a> ) foi seleccionado e correspondiam aos sinais positivos após todo processo de normalização de microarray (Passo 50,5) a partir do dia 7 gene embriões perfil de expressão bovina. Limite sinal positivo foi calculado com base na publicação permeável 16 da intensidade do sinal de um valor A. <ol><li> Para realizar a análise funcional com PANTHER (análise de proteínas através de relações evolucionárias) Classification System 23, 24, abra o seguinte link (http://www.pantherdb.org/about.jsp). </li><li> Carregar uma lista de 6.765 GS específicas do embrião para identificar processo biológico específico durante o desenvolvimento embrionário por meio do teste sobre-representação estatística e usar a configuração padrão do Bos taurus (todos os genes no banco de dados), como lista de referência 25. NOTA: Este permite a identificação de processos de desenvolvimento relacionadas com a de os termos GO que são estatisticamente super ou sub-expresso utilizando um teste binomial. </li><li> Definir limite P-valor em &lt;0,05 como padrão de software. Os dados podem ser baixados e exported em arquivo de Excel para posterior selecção 16. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Reprogramming.">Nuclear Reprogramming.</a> Studying Bovine Early Embryo Transcriptome by Microarray. , (2015).</li><li>Patterson, T. A., 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=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li></ol>

The authors have nothing to disclose.

O primeiro problema a realizar análise de microarray utilizando Dia 7 embriões não é obter quantidades suficientes de ARN de alta qualidade para estudar a expressão do gene. extração de RNA fenol tradicional / clorofórmio e método de precipitação de etanol não é recomendado para o dia 7 embriões, resultando em baixo rendimento e possíveis fenol restante inibir reação de amplificação de ARN. Em vez disso, um método baseado em coluna padrão é melhor para isolar o ARN total e, em seguida, eluir o ARN ...

Perda embrionária precoce em vacas leiteiras de alta produção é um dos principais desafios para a indústria de laticínios 1, 2. Bovine tornou-se um modelo interessante para estudar o desenvolvimento pré-implantação do embrião humano devido ao seu processo de desenvolvimento semelhante 3, 4. No entanto, é necessária mais investigação para ter uma melhor compreensão sobre genes envolvidos no desenvolvimento embrionário inicial de bovinos. Depois de vinte anos desde a primeira tecnologia de microarray desenvolvido em 1995 5, o desenvolvimento da tecnologia de fabricação sonda erros de impressão reduzidos mais sofisticados e variabilidade de chips de matriz dentro e entre diferentes plataformas de microarray 6. Tecnologia de microarray melhorou amplamente resultou numa aplicação desta tecnologia em investigação clínica 7 e, mais recentemente, no embrião inicial Qavaliação ualidade 8. A grande quantidade de material necessário para a tecnologia de microarray é a principal razão pela qual a tecnologia de microarrays inicialmente não conseguiu introduzir um número de campos de pesquisa, como o desenvolvimento embrionário inicial. Mais recentemente, métodos de amplificação de ARN foram melhorados para amplificar ARN linearmente até ao nível de sub-microgramas nanograma material de partida ARN 9. Existem vários kits de amplificação de ARN comerciais disponíveis no mercado; no entanto, os mais populares kits bem desenvolvidos estão relacionados com Ribo-Single Primer isotérmico amplificação 10 e promotor T7 conduzida 11 métodos. O mais popular de amplificação anti-sentido de ARN utiliza na transcrição in vitro com um iniciador oligo dT de ligação a um promotor de T7 na extremidade 5 &#39;12. Esta tecnologia permite a manutenção da maior parte dos transcritos anti-sentido representativas após amplificação linear fou matrizes de hibridação 13. Este método foi adaptado para amplificar nível picograma de RNA total extraído de embriões de bovino 8. Universal Linkage System (ULS) é o método de marcação que incorpora diretamente o DNA ou RNA amplificado com corante fluorescente ligada à platina quer Cyanine 547 ou Cyanine 647, formando uma ligação de coordenação sobre a posição N7 de guanina 14. Este método foi adaptado em investigação embriões para gerar mais estável, sem modificação ARNA amplificado em comparação com o aminoallyl ARNA modificada gerado pelo método enzimático 15. tanto métodos de corante único e rotulagem dois corantes foram adaptados usando Universal sistema de ligação em microarray. Um grande estudo comparações microarray foi que há uma boa correlação da qualidade dos dados entre as plataformas de matriz de um e dois de cor 6. Recentemente, tanto unidade promotor T7métodos de amplificação N ARN anti-sentido e de rotulagem ULS têm sido desenvolvidos para proporcionar um protocolo mais fiável para gerar uma quantidade suficiente de materiais de alta qualidade marcado arna para hibridação microarray 8, 16. Portanto, este estudo fornece um protocolo para demonstrar algumas das etapas importantes de extração de RNA para análise de dados envolvidos em microarray de duas cores utilizando Dia 7 embriões bovinos como um exemplo.

<table><tbody><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems</td><td>KIT0204</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent Technologies</td><td>5067-1513</td><td></td></tr><tr><td>Arcturus RiboAmp HS PLUS Kit</td><td>Applied Biosystems</td><td>KIT0505</td><td></td></tr><tr><td>2100 Bioanalyzer Instruments</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>RNA Screen Tape</td><td>Agilent Technologies</td><td>5067-5576</td><td></td></tr><tr><td>ULS Fluorescent Labeling Kit</td><td>Kreatech Diagnostics</td><td>EA-021</td><td></td></tr><tr><td>Custom Gene Expression Microarrays</td><td>Agilent Technologies</td><td>G2514F</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 1</td><td>Agilent Technologies</td><td>Part #5188-5325</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 2</td><td>Agilent Technologies</td><td>Part #5188-5326</td><td></td></tr><tr><td>2x Hi-RPM Hybridization buffer</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5190-0403</td><td></td></tr><tr><td>25x Fragment buffer</td><td>Agilent Technologies</td><td>Part #5185-5974</td><td></td></tr><tr><td>10x GE Blocking Agent</td><td>Agilent Technologies</td><td>Part #5188-5281</td><td></td></tr><tr><td>Stabilization and drying solution</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5185-5979</td><td></td></tr><tr><td>Gasket slides enabled by Agilent SureHyb techonolgy</td><td>Agilent Technologies</td><td>G2524-60012</td><td>Pack of 20 gasket slides, 4 microarrays/slide</td></tr><tr><td>Two-Color RNA Spike-In Kit</td><td>Agilent Technologies</td><td>&amp;nbsp;Cat# 5188-5279</td><td></td></tr><tr><td>GenePix 4000B array scanner</td><td>Molecular Devices</td><td>GENEPIX 4000B-U</td><td></td></tr><tr><td>Ozone Free Box</td><td>BioTray</td><td>OFB_100x200</td><td></td></tr><tr><td>GAL file</td><td>Agilent Technologies</td><td>-</td><td></td></tr></tbody></table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Um resultado representativo de ARN total e amplificado ARNA desde o dia 7 embriões é mostrada na Figura 3 e resumidos no Quadro 1. integridade do RNA e do perfil pode ser avaliada após a extração do RNA. Avaliação da qualidade do ARN podia ser realizado por instrumento Bioanalyzer (Figura 3A) e apenas as amostras com um valor superior a 7,0 RIN estão qualificados para ...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptoma Profiling de<em&gt; In-Vivo</em&gt; embriões produzidos pré-implantação dos bovinos por meio de duas cores Plataforma Microarray

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

transcriptome-profiling-vivo-produced-bovine-pre-implantation-embryos

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

7.7K visualizações.  University of Alberta. Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Vídeo: Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Expressão de proteínas fluorescentes em<em&gt; Branchiostoma lanceolatum</em&gt; Por injecção de mRNA em oócitos não fertilizados

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Biologia do Desenvolvimento

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Citoplasmática microinjeção no gado zigotos assistida laser

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Genética

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

Precisas e Fenol DNA Sexagem gratuito do dia 30 de Porcina embriões por PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonagem Funcional Utilizando um<em&gt; Xenopus</em&gt; Oocyte Sistema de Expressão

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Análise quantitativa da expressão da proteína para estudar Especificação Lineage no mouse Preimplantation Embriões

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Deficitária e Abordagem de ganho de função para Investigar celular precoce Destino Determinantes na pré-implantação embriões de camundongos

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Um método de imagem semi-alta-fonte e um kit de ferramentas de visualização de dados para analisar o desenvolvimento embrionário de C. elegans

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Análise multiplexada do sequenciamento de mRNA celular único de células embrionárias de camundongos

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Definindo o Programa de Tradução materna mRNA durante a maturação in vitro usando um ensaio de repórter oócito único

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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