Somos gratos a Nancy Papalopulu e Bonev Boyan de assistência com eletroporação Xenopus. Agradecemos também a Marc Dionne para a leitura crítica, Green Jeremy e John Wallingford para discussões úteis e os membros do laboratório de Liu pelo seu apoio. Este trabalho foi financiado por concessões do BBSRC (BB/E013872/1) e do Wellcome Trust (081880/Z/06/Z) para KJL.

Parte 1A. Equipamento Microscópio: escopo estéreo dissecar ereta com o objectivo de baixa potência <ul><li> Tensão / Gerador de Pulso: BTX ECM 830 Sistema de Eletroporação Onda Quadrada </li><li> Extrator da pipeta: P-87 Micropipeta Puller (Sutter Instrument Company, CA) </li><li> Manipulator: grossa, ou combinado grosso e fino dependendo da preparação. </li><li> Micropipeta titular: Ferramentas Ciência Belas </li><li> Eletrodo: homemade </li><li> Eletroporação câmara: homemade </li></ul> Eletrodos em forma de L: <ol><li> Corte 8 cm de alta pureza fio de tungstênio 0,4 mm (Goodfellow) e apor no ponto médio de uma seringa de 1ml utilizando putty (usamos Blu-Tack). Deixe a 4 fios de tungstênio centímetros expostos a partir da ponta da seringa e ponta curva em forma de L, 1 cm de ponta (Fig. 1A). </li><li> Apare a ponta de modo que o fim de medidas de 0,5 mm de comprimento. Tsua dica é o término do eletrodo. </li><li> Correm paralelas excesso de fio de tungstênio para seringa e usá-lo para ligar o gerador de pulso eletrodo. </li><li> Repita o processo fazendo um par de eletrodos. </li><li> Anexar eletrodos ao gerador de pulso de onda quadrada através de cabos DC. </li></ol> Eletroporação câmara <ol><li> Linha de fundo de 90 mm com prato ~ 5 plasticina non-toxic mm. </li><li> Preencha prato com media eletroporação. </li><li> Usando No. 5 relojoeiro pinça de esculpir um poço em forma de T (Fig. 2). O bem longa deve medir ~ 2 mm x 2 mm x 10 mm e a curto ~ 2 mm x 2 mm x 5 mm. O prato eletroporação pode ser lavado e reutilizado. </li></ol> 1B parte. Reagentes DNA ou macromoléculas cobrado <ul><li> Micropipetas: 1 mm de largura 4 &quot;de comprimento vidro borosilicato capilares (WPI, TW100-F) </li><li> Cultura da mídia: Normal Amphibian Media (NAM) </li><li> &gt; 10 ações X: 1100 mM NaCl, 20mM KCL, 10 mM Ca (NO 3) 2 4H • 2 O, 1 mM EDTA. <ul><li> Autoclave e armazenar a 4 ° C. </li><li> 1 X NAM: Diluir a partir de 10x estoque regulador, com 0,1 mM NaHCO 3 e 0,2 mM Na 3 PO 4. </li></ul></li><li> Xenopus laevis girinos, estágio 28 </li></ul> Preparação de DNA: <ol><li> Prepare plasmídeos de expressão, utilizando protocolos padrão. </li><li> Ressuspender o DNA para uma concentração final de 1 mg / mL em nuclease livre H 2 0. </li></ol> * Temos tido sucesso com vetores contendo um promotor CMV fortes, como pCS2 + [16]. Para a análise de linhagem, que geralmente incluem DNA de codificação de uma proteína fluorescente verde (GFP pCS2 +) em uma concentração final de 0,1 g / mL. Concentrações [DNA entre 0,1-3 mg / mL também foram testados. Descobrimos que as concentrações abaixo de 0,8 mcg / mL células ineficiente rotulados, enquanto que as concentrações de DNA maior que 2 mg / m &amp;u; l não melhorar a eficiência eletroporação]. Morfolino preparação de oligonucleotídeos: (Nota:. MOs precisam ser fluoresceinated (3&#39;-carboxifluoresceína modificada) ou carregada) <ol><li> Oligonucleotídeos ressuspender morfolino (MOs) (Genetools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ) a uma concentração de 2 mM em nuclease livre H 2 0. </li><li> Alíquota da solução de reserva de calor a 65 ° C por 5 minutos. </li><li> Diluir a concentração final de 0,5 mM em água livre de nuclease. </li></ol> * 0.1-1mM MO soluções foram testadas. 0,5 mM soluções MO foram suficientes para eletroporação de muitas células mesenquimais. Micropipetas <ol><li> Prepare micropipetas de vidro borosilicato capilares (1 mm de largura, 4 &quot;de comprimento, WPI não. TW100-F). Use extrator de agulhas para preparar micropipetas com um cone 12/08 mm de comprimento e ponta fina. </li><li> Ponta esmagar ~ mm 2da ponta com a pinça, criando uma ruptura irregulares. </li></ol> Mídia <ol><li> Meio de incubação: Prepare fresco 04/03 normal Amphibian Media (NAM) de 1x estoque. Adicionar 0,025 mg / ml de gentamicina. </li><li> Eletroporação media: como acima, com 0,1% benzocaína (Sigma, 06950). </li></ol> 2. Eletroporação Micropipeta configuração <ol><li> Preencha micropipeta com ~ 1 ml solução injectável. </li><li> Micropipeta segura em micromanipulador e anexar a microinjetor (Picospritzer II). </li><li> Micropipeta ângulo de 50 ° a partir da mesa. </li><li> Ajuste a pressão de injeção de 20 PSI. </li><li> Calibrar micropipeta para injetar 30 nl por pulso. </li></ol> Preparação de girino <ol><li> Anestesiar estágio 28 larvas por incubação de Xenopus na mídia eletroporação por 5 minutos. </li><li> Transferência de girino anestesiados em câmara de eletroporação cheia de mídia eletroporação. Embrião posiçãodentro do prazo também para que a cabeça repousa no entroncamento com a face dorsal baixo e de lado ventral exposta. A cabeça deve ser ligeiramente elevada em comparação com a cauda. </li><li> Utilizando uma pinça, gentilmente segura girino bem com em torno de plasticina. (Nota:.. Se o girino não é garantido, pode se contorcer e contato do eletrodo durante a eletroporação Neste caso, descartar o girino como tecidos faciais será severamente danificado) </li></ol> Eletroporação <ol><li> Introduza a ponta micropipeta imediatamente posterior à glândula de cimento e em mesênquima facial. </li><li> Injetar 30 nl solução em mesênquima. </li><li> Retrair micropipeta. </li><li> Alinhar rapidamente dicas eletrodo paralelo à cabeça do embrião (Fig. 3). </li><li> Aplicar 8 50 ms pulsos, 20mV quadrados. </li><li> Retrair eletrodos. </li><li> Utilizando uma pinça cuidadosamente liberação de girinos de bem e transfira para 04/03 NAM, 0.025 gentamicina mg / ml. </li><li> Girinos podem ser incubadas em 04/03 NAM, 0,025 mg / ml overnight, ou mais. </li><li> Embriões tela para eletroporação eficiente por microscopia de fluorescência, após 24 horas. </li></ol> 3. Resultados representativos: O uso de moléculas fluorescentes permite a triagem de embriões electroporated fácil. A Figura 4 mostra um lote típico de MO electroporated girinos ~ 12, 48 e 96 horas após a eletroporação, incubados a 14,5 ° C. Usando microscopia de fluorescência, MOs podem ser visualizados imediatamente após eletroporação e persistir por vários dias após a eletroporação. Em nossa experiência, a fluorescência é fracamente evidente na fase 46 (~ 5 dias depois). Nas cartilagens, fluorescência diminui drasticamente após o início da diferenciação (~ 42 º), no entanto, MO fluorescência persiste mais fortemente em outros tipos celulares, como a endoderme da faringe. Microscopia de fluorescência mostra que oligonucleotídeos são incorporados em vários tecidos craniofacial, incluindo cartilagem. Oligonucleotídeo fluorescência cmuitas vezes um ser visualizado no tecido de cada lado da cabeça. Este é provavelmente devido a rápida difusão da solução injectável em todo o mesênquima frouxo craniofacial antes de eletroporação. <img alt="Figura 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Figura 1 Homemade eletrodos. L-shaped fio de tungstênio é anexado a uma seringa de 1 ml usando non-toxic da argila ou massa de vidraceiro. (A) O término do eletrodo mede 5 mm. (B) Anexar um par de eletrodos, de modo que os términos correm paralelas. Eletrodos são ligados ao gerador de pulsos por cabos DC. <img alt="Figura 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Figura 2 câmara de eletroporação. (A) 90 milímetros prato forrado com plasticina é preenchido com a mídia e uma câmara em forma de T esculpida com uma pinça Não é relojoeiro 5. (B) O lado mais comprido mede 2 mm X 2 milímetros X10 milímetroenquanto as medidas de curto 2 milímetros X 2 mm X 5 mm. A cabeça do embrião repousa no entroncamento, parte ventral para cima. <img alt="Figura 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Figura 3 Esquema ilustrando procedimento eletroporação. St. 28 de girinos é colocado na câmara de eletroporação, lado ventral para cima. Micropipeta é inserida em glândula de cimento facial mesênquima subjacente. Injetar. Micropipeta é removido e em forma de L eletrodos são alinhados em paralelo de acompanhamento da cabeça. Aplicar oito de 50 ms, 20 pulsos mV quadrados. Retrair eletrodos. Girinos crescem para estágios desejado. Visualize MOs ou GFP expressão usando microscopia de fluorescência. <img alt="Figura 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Figura 4 girinos Representante 12 (A), 48 (B), e 96 (C) horas após eletroporação (etapas 30, 34 e 44 respectivamente). (A &quot;-B&quot;) Fluorescente MO pode ser visualizado no mesênquima craniofacial em stages 30 e 34. Fluorescência pode ser detectada nas cartilagens na fase 44 (ponta de seta,&#39;c C &quot;). O intestino é altamente autofluorescent.

<ol>
	<li>Bonev, B., Pisco, A., Papalopulu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-9+reveals+regional+diversity+of+neural+progenitors+along+the+anterior-posterior+axis.">MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis.</a> Dev. Cell. 20, 19-32 (2011).</li><li>Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+post-implantation+mouse+embryos+using+directional+electroporation+and+whole+embryo+culture.">Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.</a> Differentiation. 72, 92-102 (2004).</li><li>Drinjakovic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3+ligase+Nedd4+promotes+axon+branching+by+downregulating+PTEN.">E3 ligase Nedd4 promotes axon branching by downregulating PTEN.</a> Neuron. 65, 341-357 (2010).</li><li>Falk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+cDNA/Morpholinos+to+targeted+areas+of+embryonic+CNS+in+Xenopus.">Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.</a> BMC. Dev. Biol. 7, 107-107 (2007).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Electroporation+in+vivo+within+the+Intact+Developing+Brain.">Single Cell Electroporation in vivo within the Intact Developing Brain.</a> J. Vis. Exp. (17), e705-e705 (2008).</li><li>Kuriyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tsukushi+controls+ectodermal+patterning+and+neural+crest+specification+in+Xenopus+by+direct+regulation.">Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation.</a> of BMP4 and X-delta-1 activity. Development. 133, 75-88 (2006).</li><li>Mende, M., Christophorou, N. A., Streit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+effective+gene+knock-down+in+early+chick+embryos+using+morpholinos+but+not+pRFPRNAi+vectors.">Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors.</a> Mech. Dev. 125, 947-962 (2008).</li><li>Neumann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> Embo. J. 1, 841-845 (1982).</li><li>Price, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+motor+neuron+pool+sorting+by+differential+expression+of+type+II+cadherins.">Regulation of motor neuron pool sorting by differential expression of type II cadherins.</a> Cell. 109, 205-216 (2002).</li><li>Grossin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+gene+transfer+into+rat+articular+cartilage+by+in+vivo+electroporation.">Direct gene transfer into rat articular cartilage by in vivo electroporation.</a> Faseb. J. 17, 829-835 (2003).</li><li>Khoury, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+intra-articular+versus+systemic+gene+electrotransfer+in+experimental+arthritis.">A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis.</a> J. Gene. Med. 8, 1027-1036 (2006).</li><li>Takahashi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+cathepsin+K+in+synovium+leads+to+progression+of+osteoarthritis+in+rabbits.">Down-regulation of cathepsin K in synovium leads to progression of osteoarthritis in rabbits.</a> Arthritis. Rheum. 60, 2372-2380 (2009).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis: A Laboratory Manual. , (2000).</li><li>Wheeler, G. N., Brandli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+vertebrate+models+for+chemical+genetics+and+drug+discovery+screens:+lessons+from+zebrafish+and+Xenopus.">Simple vertebrate models for chemical genetics and drug discovery screens: lessons from zebrafish and Xenopus.</a> Dev. Dyn. 238, 1287-1308 (2009).</li><li>Turner, D. L., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+achaete-scute+homolog+3+in+Xenopus+embryos+converts+ectodermal+cells+to+a+neural+fate.">Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.</a> Genes. Dev. 8, 1434-1447 (1994).</li></ol>

Os autores não têm conflito de interesses.

Neste vídeo, demonstramos a viabilidade de eletroporação mediada entrega do gene para o mesênquima facial de Xenopus girinos. Usando essa abordagem, podemos contornar primeiros efeitos no desenvolvimento de manipular a função do gene que nos permite atingir tecidos específicos em momento posterior. Nossos estudos mostram que as populações heterogêneas de células mesenquimais craniofacial pode ser afetado, o que nos permite analisar a linhagem de células electroporated, bem como requisitos de células autônomas para as proteínas de interesse. Combinado com imagens ao vivo, podemos usar essa abordagem para estudar a função do gene, ao longo do tempo, durante o desenvolvimento craniofacial. Esse novo método destaca a rastreabilidade de Xenopus para o estudo da organogênese. Nós antecipamos que este método pode ser amplamente adaptados para estudar a morfogênese e diferenciação de outros tecidos também.

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

Eletroporação é um método eficiente de entrega de DNA e outras macromoléculas cobrados nos tecidos em momentos precisos e em locais precisos. Por exemplo, eletroporação tem sido utilizado com grande sucesso para estudar o desenvolvimento neural e da retina em Xenopus, frango e mouse 1-10. No entanto, é importante notar que em todos esses estudos, os investigadores não foram alvo de tecidos moles. Porque estamos interessados ​​no desenvolvimento craniofacial, adaptamos um método para alvejar mesênquima facial. Quando buscamos a literatura, encontramos, para nossa surpresa, poucos relatos de transferência de genes de sucesso no tecido cartilaginoso. A maioria destes estudos foram os estudos de terapia genética, como siRNA ou proteína entrega em linhagens de células condrogênicos, ou, modelos animais de artrite 11-13. Em outros sistemas, tais como a galinha ou o mouse, eletroporação de mesênquima facial tem sido um desafio (pessoal comunicandoíons, Departamento de Desenvolvimento Craniofacial, KCL). Nossa hipótese é que eletroporação em tecidos procartilaginous e cartilaginosa em Xenopus poderia funcionar melhor. Em nossos estudos, nós mostramos que a transferência de genes nas cartilagens faciais ocorre de forma eficiente nas fases iniciais (28), quando o primórdio facial ainda é composta por tecido mole antes da diferenciação da cartilagem. Xenopus é um sistema muito acessível vertebrados e para análise do desenvolvimento craniofacial. Estruturas craniofaciais são mais facilmente visível em Xenopus do que em qualquer outro modelo de vertebrados, principalmente porque os embriões Xenopus são fertilizados externamente, permitindo análises das primeiras etapas, e facilitando a imagem ao vivo na resolução única célula, assim como a reutilização das 14 mães. Entre os modelos de desenvolvimento de vertebrados externamente, Xenopus é mais útil para a análise craniofacial de peixe-zebra, como larvas Xenopus são maiores e mais fácil de dissect, ea região em desenvolvimento facial é mais acessível à imagem do que a região equivalente em peixe. Além disso, Xenopus é evolutivamente mais próximos do homem do que peixe-zebra (~ 100 milhões ano mais próximo) 15. Finalmente, nestes estágios, girinos Xenopus são transparentes, e expressão simultânea de proteínas fluorescentes ou moléculas permitirá uma fácil visualização das cartilagens em desenvolvimento. Nós antecipamos que esta abordagem irá permitir-nos rapidamente e eficientemente teste moléculas candidato em um sistema modelo in vivo.

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

Cartilagens craniofacial desenvolver em estreito contato com outros tecidos e são difíceis de manipular em animais vivos. Estamos usando eletroporação para entregar ferramentas moleculares durante o crescimento do esqueleto craniofacial, enquanto ignorando cedo efeitos embrionárias. Esta abordagem irá permitir-nos testar de forma eficiente as moléculas de candidato<em&gt; In vivo.</em

Eletroporação de Mesênquima Craniofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K visualizações.  King's College London. Cartilagens craniofacial desenvolver em estreito contato com outros tecidos e são difíceis de manipular em animais vivos. Estamos usando eletroporação para entregar ferramentas moleculares durante o crescimento do esqueleto craniofacial, enquanto ignorando cedo efeitos embrionárias. Esta abordagem irá permitir-nos testar de forma eficiente as moléculas de candidato In vivo.

Vídeo: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Eletroporação de Micobactérias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Biologia

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Avaliar Propriedades Sinalização de Ectodérmica Epitélio Durante o Desenvolvimento Craniofacial

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Um ensaio de explante para avaliar o comportamento celular do mesênquima craniana

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Do genoma deleções de genes em<em&gt; Streptococcus sanguinis</em&gt; Por PCR de Alto Rendimento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

Separação de Ectoderma embrionárias de camundongos Facial e mesênquima

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualização de Desenvolvimento Craniofacial nas SOX10: Kaede Transgênicos Zebrafish Linha Usando Time-lapse microscopia confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analisando Craniofacial morfogênese em Zebrafish Usando 4D microscopia confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para pluripotentes estaminais humanas produção de células, Manutenção e Análise Genética

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Avaliando Contribuições Espécies específicas Para o Desenvolvimento Craniofacial Utilizando Quimeras Codorniz-pato

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...