Os autores gostariam de agradecer a Irene Choi para ilustrar cabeças de embriões em Fig. 1 e os outros membros do laboratório de ajuda e discussão. Este trabalho é apoiado pelo NIH concessão DE012728 (TW)

1. Prepare Dispase II <ol><li> Preparar uma solução fresca de Hepes-solução salina tamponada (HBS) (50 mM de Hepes / KOH, pH 7,4, 150 mM NaCl). Adicionar 2 ml de 0,5 M de tampão Hepes / KOH a pH 7,4 e 1,2 ml de 2,5 M de NaCl em 16,8 ml de H 2 O. Ultrapure </li><li> Faça 10 mg / ml Dispase II. Pesar 0,2 g Dispase II (Roche, Cat. # 4942078001) e dissolver em 20 ml de HBS. Preparar 1,0 ml alíquotas em tubos de Eppendorf individuais, e armazenar a -20 ° C para armazenamento a longo prazo. </li><li> Diluir 10 mg / ml Dispase II 1:10 em PBS antes da utilização. Coloque a Dispase II diluído em gelo. </li></ol> 2. Dissecar CD-1 embriões E10.5 de mulher grávida Todos os experimentos com animais foram realizados de acordo com os protocolos aprovados pelo Animal Care Universidade do Colorado, Denver (UCD) e Comitê de uso. Ratinhos CD-1 (ICR) foram obtidos a partir de Harlan Laboratories (Indianapolis, IN). <ol><li> Limpe a área de dissecção e ferramentas com etanol 70%. </li><li> Euthanize fêmea CD-1 grávidas usando aprovaprotocolos ed. </li><li> Pulverizar o abdômen com etanol 70%, em seguida abrir o abdômen. Usar um conjunto de pinças para retirar o útero e outro para rasgar o mesométrio do útero. Remover o útero do corpo. Repita o procedimento para corno uterino no outro lado do abdômen. </li><li> Resumidamente enxaguar o útero com PBS gelado em uma placa de Petri de 10 cm, para lavar o sangue. </li><li> Transferir o útero para uma nova placa de Petri com PBS gelado. Separar em um embrião de-fragmentos cortando entre os locais de implantação. </li><li> Transferir um embrião em PBS gelado sob um estereomicroscópio. Use uma pinça fina para arrancar a parede muscular do útero a partir de um dos locais de corte para expor o embrião com o saco vitelino. Retire o saco vitelino e âmnio. A transferência de embriões dissecados para uma placa de Petri de 6 cm com PBS em gelo. Prossiga até que todos os embriões foram coletados desta forma. </li></ol> 3. Dispase II Tratamento <ol><li> Lave todos os embriões com PBS numa placa de Petri de 6 cm. SubstituirPBS com 10 ml diluídos em PBS Dispase II. Cobrir a placa de Petri e incubar a 37 ° C durante 25 minutos - uma incubadora bacteriana ou sala quente deve ser suficiente - não ter usado um tecido de CO 2 incubadora de cultura para esta etapa. </li><li> Pegue a placa de Petri para fora e observar o embrião sob estereomicroscópio. O ectoderma deve ter se soltado, caracterizado por uma diferença clara entre esta camada de tecido e no mesênquima subjacente. Se não, incubar por mais 5 min a 37 ° C. Em seguida, coloque a placa de Petri no gelo. </li></ol> 4. Dissecar os destaques intactas Facial <ol><li> Use uma pipeta de vidro descartável (VWR, Cat # 14672-380) para transferir um Dispase II tratados embrião em PBS gelado em uma placa de Petri de 6 cm sob um estereomicroscópio. </li><li> Utilizar um par de pinças para segurar o embrião. Utilizar um outro par de fórceps finos cuidadosamente para cortar os limites das proeminências faciais associadas a cabeça (Figura 1 mostra os limites daos destaques facial). Iniciar a partir de um lado da MnP, em seguida, o MxP e FNP o último. Fazer o mesmo para o outro lado para dissecar as proeminências para fora da face intacto (Figura 2A). </li></ol> 5. Ectoderma Facial separado do mesênquima <ol><li> Observar as proeminências faciais sob um estereomicroscópio com distância de trabalho de 3x. Use um longo vidro Pasteur pipeta suavemente para pipetar as proeminências faciais cima e para baixo 5 vezes. Depois disso, a ectoderme é fácil de descascar. </li><li> Use uma pinça para segurar as proeminências faciais. Usar o outro par de pinças para retirar a ectoderme muito lentamente. Figura 2B mostra as folhas separadas de ectoderma facial. </li><li> Usar uma nova longo vidro Pasteur pipeta para transferir o ectoderma facial em um tubo Eppendorf de 1,5 ml com PBS em gelo. Transferir o mesênquima para outro tubo Eppendorf de 1,5 ml com PBS em gelo. </li><li> Use o novo gelada PBS para cada dissecção. Coletar amostras. </li><li> Lave tele amostras com PBS. Centrifugar a 4 ° C, 500 xg durante 3 min. </li><li> Aspirar o PBS utilizando pipetas de vidro longos de Pasteur. Se as amostras devem ser usados ​​para o ensaio de chip, vá para o passo 6.1. Para a extração de RNA, vá para o passo 6.2. Para a extração de proteínas, vá para o passo 6.3. </li></ol> 6. Amostras de processo para novas experiências <ol><li> As amostras para ensaio ChIP <ol><li> Adicionar 1 ml de PBS com formaldeído a 1% e de reticulação. Homogeneizar as amostras. </li><li> Incubar à temperatura ambiente durante 10 minutos com rotação. </li><li> Adicionar 110 ul de 1,25 M de glicina a cada tubo para extinguir o formaldeído que não reagiu. </li><li> Misturar e incubar à temperatura ambiente durante 5 min. </li><li> Girar a 500 xg a 4 ° C durante 3 min. Remover o sobrenadante. </li><li> Adicionar 1 ml de PBS frio para lavar tecidos. </li><li> Girar a 500 xg a 4 ° C durante 3 min. </li><li> Remover PBS e PBS lavar repetir uma vez. </li><li> Remover o sobrenadante, Encaixe congelar os tecidos em azoto líquido. Armazenar a samples a -80 ° C para posterior partilha e processamento. </li></ol></li><li> As amostras para a extração de RNA <ol><li> Coloque o ectoderma facial e mesênquima em tubos contendo RNAlater (Ambion). </li><li> Incubar as amostras em RNAlater solução durante a noite a 4 ° C para permitir a penetração profunda do tecido. </li><li> Armazenar a -20 ° C para subsequente conjugação e processamento. </li></ol></li><li> As amostras para a extração de proteínas </li></ol> Adicionar tampão de lise para a amostra para o processamento subsequente. Ou tirar congelar os tecidos em nitrogênio líquido. Armazenar as amostras a -80 ° C para subsequente conjugação e processamento. Notas <ol><li> Limitações de tempo. Pensando em 4-6 horas desde o início da dissecção até o início da seção 6. </li><li> Quando proficiente, leva ~ 10-15 minutos por embrião para realizar seções 4 e 5 (dissecção destaque e separação ectoderme). </li><li> Se as amostras são para o isolamento de ARN, cada solution deve ser tratado com dietilpirocarbonato primeiro. Também limpar a pinça e da área de trabalho com RNase Fora. </li><li> Mantenha as amostras em gelo em todos os momentos, exceto para o tratamento II Dispase e dissecações. Mudar para nova gelada PBS para cada dissecção para garantir que o PBS utilizado para dissecção é frio. </li><li> Pinças cortantes são cruciais para obter a dissecção precisa. Limpe a pinça completamente para evitar qualquer contaminação. </li><li> Para a coleta de amostras de proeminências diferentes, dissecar cada proeminência separadamente após Dispase tratamento II. Em seguida, separar o ectoderma e mesênquima. </li><li> Após o tratamento com Dispase II, o ectoderma facial é fácil de descascar. Preste atenção especial à contaminação por ectoderma outro. Descartar todo o tecido potencialmente contaminadas. </li><li> Depois de começar, não pare até que as amostras atingir o ponto adequado na Secção 6. Concluir separação de ectoderma facial e mesênquima para todos os embriões tão rapidamente como você pode. </li><li> O e-atividades nzyme de Dispase II pode ser diferente entre os lotes e marcas. É necessário regular o tempo de tratamento para as diferentes marcas e lotes de Dispase II. </li></ol>

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., 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=The+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., 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=The+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

Os autores não têm interesses conflitantes ou outros conflitos associados com o conteúdo deste artigo.

Este protocolo fornece um método simples para separar ectoderma rato embrionário mesênquima facial e base em uma primeira etapa de tratamento Dispase II. Em estudos anteriores, temos realizado dissecção das proeminências faciais antes Dispase tratamento II, mas temos de forma consistente que o mesênquima se torna &quot;pegajoso&quot; e mais difícil de manipular. Nosso novo protocolo evita esse problema. Combinação Dispase II tratamento com força física suave pipetando para cima e para baixo faz a separação...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

Fendas orofaciais são os defeitos mais freqüentes craniofaciais, que afetam 1,5 em 1000 1,2 recém-nascidos em todo o mundo. Fissuras orofaciais é causada por três desenvolvimento anormal facial. No ser humano e do rato, o crescimento inicial e padronização da cara depende de várias papilas pequenas de tecido, as proeminências faciais 4,5. O rosto é derivado de seis principais destaques: emparelhados processos frontais nasais (FNP), proeminências maxilares (MXP) e proeminências mandibulares (MDP). Estes consistem de proeminências inchaços de mesênquima que estão incorporados em um epitélio de revestimento. Estudos realizados em várias espécies têm mostrado que a sinalização crosstalk entre ectoderma facial e mesênquima é crítico para a formação da face 6. No entanto, os detalhes mecanísticos sobre os genes envolvidos nestes relés de sinalização são escassos. Uma maneira de obter uma compreensão abrangente da expressão do gene, fator de transcrição de ligação, e marcas de cromatina associados ao desenectoderme pingue facial e mesênquima é isolar e caracterizar os compartimentos dos tecidos separados. Aqui apresentamos um método para separar ectoderma facial e mesênquima no dia embrionário (E) 10,5, uma fase crítica de desenvolvimento na formação do mouse facial que precede a fusão dos destaques. O nosso método é adaptado a partir do método já anteriormente utilizado para dissecar proeminências faciais 7. Neste estudo anterior havia sido empregado C57BL inato / 6 ratos como esta cepa se tornou um padrão para a genética, morfologia facial genômica e 8. Aqui, no entanto, devido às quantidades mais limitadas de tecido disponível, nós utilizamos a estirpe CD-1 outbred que é mais barata para comprar, mais robusto para a criação, e que tende a produzir mais embriões (12-18) por ninhada do que qualquer inbred rato tensão 8. Seguindo o isolamento do embrião, neutro protease Dispase II foi usada para tratar o embrião inteiro. Em seguida, as proeminências faciais foram dissecared para fora, e a ectoderme facial foi separado do mesênquima. Este método mantém tanto ectoderme facial e mesênquima intacta. As amostras obtidas utilizando este método pode ser usado para a detecção de proteínas, incluindo as técnicas, a imunoprecipitação da cromatina (ChIP) ensaio, os estudos de microarranjos e RNA-seq.

Após tratamento Dispase II, a ectoderme do embrião tende a ser solto. As proeminências faciais devem estar intacto após dissecção (Figura 2A). O ectoderma facial isolada é clara e livre de mesênquima tecido (Figura 2B). Para determinar a eficácia do protocolo e para detectar a contaminação cruzada nós ensaiadas para prosencéfalo expressaram Zic3 9, ectodérmica específico CDH1 5 e mesenquimal específico SOX10 10 utilizando transcriptase re...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Um protocolo para a separação da ectoderme embrionária facial e mesênquima é descrito. Usamos Dispase II para tratar embriões inteiros primeiro, dissecar todo proeminências faciais, e então separar o ectoderma facial e mesênquima.

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 ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

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Biology

10.8K visualizações.  University of Colorado Denver Anschutz Medical Campus. Um protocolo para a separação da ectoderme embrionária facial e mesênquima é descrito. Usamos Dispase II para tratar embriões inteiros primeiro, dissecar todo proeminências faciais, e então separar o ectoderma facial e mesênquima.

Vídeo: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Separação eletroforética de proteínas

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Adenovírus mediada por remoção genética das moléculas de sinalização em fibroblastos de rato cultivadas Primária embrionárias

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e cultura de células da zona nefrogênica do rim do rato embrionárias

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Translocação polarizada de proteínas fluorescentes em Xenopus Ectodérmicas em resposta à sinalização Wnt

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

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. ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Diferenciação eficiente de células-tronco embrionárias em neurônios motores

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparação de células embrionárias de camundongos Fibroblastos adequados para a cultura de células-tronco embrionárias humanas e pluripotentes induzidas

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...