Este protocolo foi desenvolvido nos laboratórios da Jane Dodd, Thomas Jessell e Lumsden Andrew. Muitas pessoas, incluindo Konrad Basler, Calof Ann, Edlund Thomas, Hamilton Phil, Karagogeos Domna, Ruiz Ariel i Altaba e Yamada Toshiya determinada como explantes cultura da medula espinhal em colágeno. Marysia Placzek e Marc Tessier-Lavigne foi pioneiro na técnica de utilização de crescimento do axônio em explantes in vitro como forma de identificar moléculas de orientação axônio. Trabalho no laboratório Butler é suportado por concessões do March of Dimes e R01 NS063999 de NIH / NINDS.

Parte 1: Preparação de agregados de células COS Transfectados usando gotas de suspensão <ol><li> COS-7 sementes (COS), as células em placas de cultura de 35 mm. Quando chegar a 80% confluência, 1μg transfecção do plasmídeo de expressão para as células usando Lipofectamine2000 acordo com o protocolo do fabricante s. </li><li> Para preparar pendurado gotas, aspire o meio de transfecção e lavar as células transfectadas COS com 1ml de 1x Phosphate Buffered Solution (PBS). Tratar células com 0,5 ml de enzima meio livre de células dissociação por 15 minutos. Adicionar 1ml de uma solução de Opti-MEM + 1x Penicilina / Estreptomicina / Glutamina (P / S / G) + 10% de soro fetal bovino (FBS) para parar a reação. </li><li> Triturar as células para removê-los da superfície da placa de cultura e transferência para um tubo cônico 15ml. Rotação por 2 minutos a 2000K para agregar as células. Remover o sobrenadante e ressuspender pellet em 100μl de Opti-MEM + 1x P / S / G + 10% FBS. </li><li> Local várias quedas de 20μl no interior de uma tampa 35 milímetros cultura prato, inverter tampa e coloque em cima do fundo do prato e deixe em 37 ° C incubadora durante várias horas até que as células agregadas. </li></ol> Parte 2: Preparação de explantes Dorsal da Medula Espinhal <ol><li> Dissecar embriões de ratos E11 do útero da mãe e manter em gelo em L15 médio até necessário. </li><li> Com uma agulha de tungstênio afiada, remover uma seção segmento 4-6 a partir do tronco de cada embrião, a partir da região imediatamente abaixo forelimb bud. Usando uma pipeta, recolher os pedaços de tecido em um poço de um prato de 4 Nunc bem e manter em gelo. </li><li> Quando todos os segmentos foram dissecados embrião, use a pipeta para transferir as peças em uma solução de 1ml L15 + 1mg dispase em um segundo poço do prato Nunc. Incubar a temperatura ambiente por mais de 5 minutos. Não mais de incubar os pedaços de tecido com dispase. </li><li> Durante a incubação, adicionar 0,5 ml de soro de cabra calor inativado normal (HIGS) para cerca de 10ml de L15 em uma placa de Petri e agite para misturar. Transferência de 1 ml desta solução em um terceiro poço do prato Nunc e transferir os pedaços de tecido para esta solução quando o período de incubação é longo. Manter no gelo. Nota: a dissecção posterior será mais fácil se os pedaços de tecido têm permissão para &quot;descansar&quot; no gelo por uma hora. </li></ol> Parte 3: Colágeno Priming <ol><li> Adicionar 40μl meio mínimo 10x essenciais ao colágeno 360μl, misture rapidamente e minuciosamente por um movimento súbito ou brevemente o tubo vortex. Girar rapidamente em uma picofuge. A solução será amarela. Mantenha no gelo, tanto quanto possível. </li><li> Adicionar o suficiente de bicarbonato de sódio 0,8 M (NaHCO 3) para ligar a solução de colágeno ligeiramente laranja (ver nota abaixo). Mais uma vez, misture rapidamente e minuciosamente por um movimento súbito e spin down brevemente em um picofuge. Se a solução continua a ser cor de rosa após a mistura, você adicionou NaHCO demais 3 e terá de começar novamente com um novo tubo de colágeno. </li><li> Neste ponto, a solução de colágeno está ferrado. Ele permanecerá líquido no gelo, mas solidifica dentro de cerca de 5 minutos (tornando-se rosa) quando à temperatura ambiente. </li><li> 20μl local de colágeno em cada poço de uma placa de quatro poços Nunc. Usando a ponta de sua pipeta, espalhar-se o colágeno para formar um &quot;bloco&quot; de pequeno porte. Deixe o colágeno fixado em temperatura ambiente. </li></ol> Nota: A quantidade exata de NaHCO 3 terão de ser ajustadas para cada lote de colágeno. Iniciar baixa (11μl) e em seguida, adicione 0,5 1μl gradual até que a solução é uma máscara leve de laranja. A quantidade &quot;certa&quot; é geralmente menos de 1ul a menor quantidade de NaHCO 3, que viraria a rosa colágeno. A quantidade de NaHCO 3 não escala linearmente para cima ou para baixo. Parte 4: Dissecção da Dorsal Explantes da Medula Espinhal e Posicionamento deles com agregados celulares COS em Colágeno Matrix <ol><li> Usando uma agulha de tungstênio recém afiada e um par de pinças finas (# 5 ou # 55), remova o mesoderma em torno da medula espinhal. </li><li> Corte paralelo à placa de fundo com a agulha de tungstênio, remova cuidadosamente o quinto ventral da medula espinhal. </li><li> Bissetriz do explante dorsal da coluna vertebral, certificando-se que as bordas são cortadas de forma limpa possível. Re-aparar as arestas, se necessário. Transferir cada explante dorsal da coluna vertebral em almofadas de colágeno separado usando uma pipeta boca equipado com um tubo de vidro capilar puxado para aprox. 0,5 milímetros de diâmetro. </li><li> Corte o agregado de células transfectadas COS em quadrados de aproximadamente a mesma largura que a borda lateral do explante dorsal da coluna vertebral. </li><li> Transferência de um desses quadrados sobre o bloco de colágeno com o explante dorsal da coluna vertebral, com uma pipeta na boca. </li><li> Com a pipeta na boca, aspirar fora de todo o excesso de líquido usando a pipeta na boca. Não mais de aspirar. </li><li> Aplicar 4μl de colágeno injetado sobre o bloco, e, utilizando a agulha de tungstênio, Mova o colágeno ao longo dos explantes diretamente sem tocar no tecido. Movendo a agulha de tungstênio no colágeno ao redor dos explantes, orientar os explantes para que o agregado de células COS é adjacente à borda lateral do explante dorsal da coluna vertebral. </li><li> Depois que o colágeno tem set, repetir a aplicação de 4μl de colágeno para garantir que os explantes são completamente envolto em colágeno. </li><li> Em uma capa de cultura de tecidos, adicionar 0,5 ml de Opti-MEM + 1xP/S/G e cultura para 30-40 horas. </li><li> Fix explantes por 45 minutos com paraformaldeído a 4% de 0,5 ml em 1xPBS, mantendo a placa de gelo. </li><li> Lave duas vezes com 0,5 ml de uma solução de bloqueio de 1xPBS + 0,1% Triton-X100 HIGS +1% (PBTN). Bloco no 4 ° C por pelo menos um par de horas, uma noite para o dia de incubação é preferível e então o processo por imuno-histoquímica. </li></ol> Parte 5: Visualização de Axônios comissural por Imunohistoquímica <ol><li> Após o bloqueio em PBTN, corte os explantes para fora da placa 4 bem com uma pinça e coloque em um prato de 48 também. </li><li> Adicionar 200μl de anticorpos primário para cada bem e deixe durante a noite a 4 ° C. Para visualizar os axônios comissurais, use uma solução 1:06 de mouse de anticorpos anti-Tag1. Para determinar se a transfecção foi bem-sucedida, também incluem um anticorpo primário contra qualquer molécula de sinalização que está sendo testado, ou o epitopo relevante se a molécula de sinalização foi epítopo marcados. </li><li> Lave cada amostra por 5 x 1 hora em PBTN a 4 ° C. </li><li> Adicionar 200μl de anticorpo secundário para cada bem e deixe durante a noite a 4 ° C. Se anti-Tag1 anticorpos foram utilizados como anticorpo primário, use uma cabra de anticorpos IgM anti secundário do mouse acoplado a qualquer FITC ou Cy3. Se estiver usando sensível à luz anticorpos secundários acoplados a FITC ou Cy3, mantenha o prato de 48 bem coberta em papel alumínio a partir deste ponto. </li><li> Lave cada amostra 5 x 1 hora cada um em PBTN a 4 ° C. </li><li> Transferência dos explantes para um slide depressão 3-bem, retire o excesso de líquido, montagem em Vectashield médio e lamela. Armazenar em uma caixa de luz de segurança em cada 4 ° C ou -20 ° C. </li></ol> Parte 6: Imagens Representante de explantes Em um experimento bem sucedido, o explante e agregar COS permanecerá ao lado um do outro e não se afastar como o colágeno sets. Haverá overgrowth mínima de axônios; overgrowth axonal significativa indica que o explante dorsal da coluna vertebral foi cultivado por muito tempo. Não haverá bolhas de crescimento do explante; blebbing ocorre se o tecido entra em contato direto com o meio de cultura. A capacidade da molécula de sinalização para reorientar axónios é avaliado através de microscopia confocal. A extensão da reorientação axônio pode ser quantificado através da medição do ângulo médio de reorientação dos axônios mais próximo à célula COS 3 agregada. Conforme mostrado na Figura 1, as células COS expressando-myc tag BMP7 (azul) reorientar Tag1 + axônios comissurais (verde) de distância da fonte de BMP7 (Figura 1B), semelhante à maneira em que um explante de chapa de teto repele axônios comissurais (Figura 1A). Células COS expressar um vetor de controle não têm efeito sobre a orientação dos axônios comissurais 3,4. Estes explantes foram também marcadas com anticorpos contra o fator de transcrição Isl1 / 2 (vermelho), que decora os neurônios motores e interneurônios dorsal. Este resultado foi a primeira indicação de que um membro da família BMP medeia a atividade dos três chapa de teto chemorepellent. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> Figura 1: Por favor, <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1.

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

Os fatores críticos que determinam o sucesso na realização deste ensaio são, primeiro, o tecido não devem ser tratados com dispase por muito prolongado período, este tratamento irá resultar no tecido tornando-se muito pegajoso e ter uma redução da viabilidade. Dois, o colágeno devem estar perfeitamente preparados e mantidos em gelo, tanto quanto possível. Vai tornar-se cada vez mais difícil lidar com se começa a definir, ou seja, passam a rosa. Três, a agulha de tungstênio devem ser mantidos sempre muito ...

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

Dorsal axônios comissurais na medula espinhal dos vertebrados 1 ter sido um modelo de sistema de valor inestimável em que identificar sinais de orientação axônio. Aqui, descrevemos um ensaio in vitro &quot;, a reorientação do ensaio&quot;, que tem sido amplamente utilizado para estudar o efeito dos sinais extrínsecos e intrínsecos na orientação dos axônios comissurais 2. Este ensaio foi desenvolvido por várias pessoas nos laboratórios da Jane Dodd, Thomas Jessell e Lumsden Andrew (ver agradecimentos para mais detalhes) e versões deste ensaio foram usadas para demonstrar as atividades reorientação de orientação das moléculas-chave axônio, incluindo o BMP chemorepellent na chapa de teto 3,4 e as atividades de quimioatratante Netrin1 5 e Sonic hedgehog (Shh) 6 na placa de fundo na medula espinhal. Explantes compreendendo 2-3 segmentos da dorsal de dois terços da medula espinhal são dissecados a partir de dia embrionário (E) 11 ratos e cultivadas em três géis de colágeno dimensional 7. E11 dorsal explantes espinhal contém recém-nascido neurônios comissurais, que podem ser identificadas pela sua expressão axonal da glicoproteína, Tag1 8. Ao longo de 30-40 horas em cultura, a trajetória dos axônios comissurais está resumida nestas explantes dorsal com um curso de tempo similar ao observado in vivo. Essa trajetória axonal pode ser contestada, colocando tecidos ou de teste ou um agregado de células COS expressando uma molécula de sinalização candidato em contato com uma das bordas laterais do explante dorsal. Axônios comissurais estendendo na vizinhança do tecido anexado vai crescer sob a influência de ambas as placas do telhado do endógenos e sinais do tecido ectópico lateral. O grau em que os axônios comissurais são reorientadas sob essas circunstâncias podem ser quantificados. Com este ensaio, é possível tanto para examinar a suficiência de um sinal especial para reorientar axônios comissurais 3,4, assim a necessidade de este sinal para direcionar a trajetória comissural 9.

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Este ensaio avalia a capacidade de uma molécula de sinalização, aqui proteína morfogenética óssea 7 (BMP7), para reorientar axônios comissurais. Um explante da medula espinhal dorsal embrionárias cultivadas é adjacente a um agregado de células secretoras de COS os fatores de crescimento candidato. Reorientou axônios comissurais crescendo dentro do explante são visualizados através da imuno-histoquímica.

Doseamento do Capacidade de difusíveis moléculas sinalizadoras para Reorientar embrionárias Spinal Axônios comissural

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Research

JoVE Journal

Biology

8.8K visualizações.  University of Southern California. Este ensaio avalia a capacidade de uma molécula de sinalização, aqui proteína morfogenética óssea 7 (BMP7), para reorientar axônios comissurais. Um explante da medula espinhal dorsal embrionárias cultivadas é adjacente a um agregado de células secretoras de COS os fatores de crescimento candidato. Reorientou axônios comissurais crescendo dentro do explante são visualizados através da imuno-histoquímica.

Vídeo: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biologia

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Eletrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura do mouse pulmão embrionário, um sistema para avaliar os mecanismos moleculares de Ramificação

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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 ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotobranqueamento Ensaios (FRAP &amp; FLIP) a Medida Dynamics Protein Chromatin em Viver Células-Tronco Embrionárias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Doseamento do atividade da quinase de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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 ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Explantes blastômero para testar Compromisso destino celular durante o desenvolvimento embrionário

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

A estimulação mecânica induzida por cálcio Propagação de Ondas em monocamadas de células: O Exemplo de Bovinos endotélio corneano

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).