Este trabalho foi financiado pelo NIH conceder NS042197 a MD. AJK foi suportada por concessões T32GM008719 e F30NS068006.

1. Produção de agulhas microinjeção <ol><li> Agulhas pré-fabricados microinjeção estão disponíveis comercialmente (eg. Femtotips de Eppendorf) e são úteis se não se está realizando um grande número de microinjeções. No entanto, para aqueles que desejam estabelecer a longo prazo as capacidades para microinjecting, uma alternativa é produzir agulhas microinjeção no laboratório usando de parede fina de vidro de borosilicato capilares e um extrator de agulhas comerciais. Isso também permite que a forma de agulhas para ser variado, que pode ser útil para diferentes tipos de células. </li><li> Com o Extrator Needle Narishige PC-10 microinjeção, anexar todos os quatro pesos e use um programa de uma etapa puxar (configuração Etapa 1) com a definição de calor em relação a 58,0 (aquecedor No.2). Certifique-se de colocar o elemento de aquecimento no centro de cada capilar para que as duas agulhas resultantes são um período similar. </li><li> Puxe capilares várias (cerca de 2 capilares para cada proteína de interesse). </li><li> Agulhas armazenar em um recipiente com a certeza de não danificar a ponta da agulha. Materiais como espuma ou Blu-Tack pode ser usado em recipientes para armazenar agulhas microinjeção. </li></ol> 2. Preparação de misturas de proteínas para Injeção <ol><li> Prepare um buffer de microinjeção 10x contendo 1 M de cloreto de potássio e fosfato de potássio 0,1 M (KPI) tampão (mistura equimolar de K 2 HPO 4 e KH 2 PO 4) para um pH de 7,4. Este buffer pode ser armazenado a longo prazo na temperatura ambiente. </li><li> Para visualizar a injeção, um corante fluorescente rodamina-dextrano, como pode ser adicionada. Diluir tampão microinjeção the10x na água e dissolver rodamina-dextran em pó para fazer uma solução tampão 5x microinjeção contendo 20-40 mg / mL rodamina dextran. </li><li> Conservar a solução no escuro a 4 ° C. Deve ser suficiente para até 100 preparações solução individual de proteínas. Outros corantes, como o isotiocianato de fluoresceína-dextran pode substituir. </li><li> Prepare stocks citocromo c purificado pela dissolução do citocromo c em água a uma concentração de 20 mg / mL. Loja do citocromo c a -80 ° C para armazenamento de longo prazo e evitar os ciclos de congelamento e descongelamento, armazenando citocromo c em pequenas (~ mL 10) alíquotas. </li><li> Prepare uma mistura de proteínas 10 mL para injeção através da combinação de buffer de 2 mL microinjeção 5x contendo rodamina-dextran com 3 mL de água e 5 mL do citocromo c para uma concentração final de 10 mg / mL do citocromo c em 1x tampão (100 mM KCl, 10 mM KP i, 4-8 mg / mL rodamina dextran). </li></ol> 3. Microinjeção citoplasmática de citocromo c <ol><li> Pouco antes de microinjeção, centrifugar a mistura do citocromo c e buffer microinjeção a 16.000 g por 10 minutos a 4 ° C para separar quaisquer partículas que podem entupir agulhas microinjeção. </li><li> Durante a centrifugação, ligue o microinjetor para permitir que a pressão do ar para construir. </li><li> Coloque um prato de células no centro do palco microscópio e definir o foco sobre as células. Para minimizar a quantidade de tempo que as células são mantidas fora da incubadora, as células são normalmente retornados para a incubadora dentro de 30 min. </li><li> Pipeta 0,5-1 mL da superfície superior da mistura de proteínas na extremidade sem corte do capilar. Tenha cuidado para não pipetar as partículas que foram centrifugados para o fundo do tubo. Dentro de um minuto, a mistura de proteína irá distribuir para a ponta da agulha através da ação capilar. </li><li> Coloque a agulha firmemente ao titular capilar do micromanipulador e posicionar a agulha de modo que sua ponta passa através da luz transmitida do microscópio em aproximadamente um ângulo de 45 °. </li><li> Ajustar a posição da ponta da agulha para que ele seja localizado, no centro do campo de visão. Para centralizar a agulha, use o micromanipulador para mover a agulha ao olhar através da ocular do microscópio. A sombra agulha deve ser visível. Ajuste a agulha de modo que sua sombra é visto apenas em uma metade do campo de visão, indicando que a ponta da agulha está centrada acima das células. </li><li> Definir o microinjetor para o modo de fluxo contínuo e definir a pressão de trabalho de 20-100 hPa. Cada agulha pode exigir uma pressão de trabalho diferente, a pressão de trabalho e provavelmente vai precisar de ajustes durante o processo de microinjeção de manter um fluxo constante. </li><li> Abaixe a agulha com o botão grossa para uma posição um pouco acima das células. Para fazer isso, elevar o plano focal do microscópio para uma posição um pouco acima das células. Depois abaixe a agulha em direção as células usando o botão grossa até a ponta da agulha está em foco. </li><li> Re centro-ponta da agulha dentro do campo de visão e aumentar a ampliação, alterando a objetiva do microscópio. </li><li> Abaixe lentamente a agulha usando o botão unti multa manipuladorl agulha é apenas ligeiramente acima do plano focal das células. </li><li> Verificar o fluxo da mistura de proteínas, olhando para a fluorescência vermelha da rodamina. A mistura de proteínas deve ser sair a agulha como um fluxo fino e constante. As agulhas devem ser substituídos e re-loaded Se a agulha estiver comprometida ou se o fluxo é muito forte. Às vezes, a ponta do capilar de vidro está fechado e sem fluxo é visto a partir da agulha. Se isso ocorrer, substitua a agulha com cuidado ou abaixá-lo para o fundo do prato cultura gentilmente ruptura da ponta da agulha. </li><li> Posição da ponta da agulha de forma que ele está apontando para uma célula em aproximadamente um ângulo de 45 °. Em seguida, com um movimento suave, abaixar a agulha enquanto movê-lo para o celular. Com um movimento suave segundo, imediatamente inverter a direção da agulha para removê-lo da cela. </li><li> Uma célula injetou com sucesso, muitas vezes, ligeiramente inchar e pode ser confirmado através da visualização da rodamina vermelho fluorescente dentro da célula. Ocasionalmente, uma célula vai ser acidentalmente injetado no núcleo, que será visível. </li><li> Continuar a injetar células, ajustando o estágio do microscópio até cerca de 50-100 células são injetadas. Ao mover o estágio do microscópio, certifique-se de levantar a agulha microinjeção de modo que ele limpa a parte superior das células. </li></ol> 4. Resultados representativos: A microinjeção citoplasmática de citocromo c imita o seu lançamento a partir da mitocôndria durante a apoptose. Assim, como esperado, fibroblastos rapidamente sofrem apoptose após microinjeção citosólica de bovinos do citocromo c (Fig. 1A). Para garantir que o procedimento de injeção por si só não é responsável pela morte celular, a injeção de levedura citocromo c serve como um controle importante, já que o fermento do citocromo c é incapaz de ativar caspases 12. Curiosamente, pós-mitóticas neurônios simpáticos são notavelmente resistentes à citosólica do citocromo c (Figura 1B) 8,13. Nosso laboratório identificou que o XIAP inibidor endógeno é um inibidor da caspase-chave de ativação do caspase em neurônios 14. Assim, para os neurônios à morte após a injeção do citocromo c, XIAP deve primeiro tornar-se desativado. Por exemplo, microinjeção de citocromo c em xiap - / - neurônios simpáticos é suficiente para permitir a ativação das caspases e apoptose nessas células (Fig. 2). <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Figura 1"/> Figura 1. Microinjeção citoplasmática de citocromo c induz a morte rápida em fibroblastos, mas não os neurônios. A) do tipo selvagem MEFs ou (B) 5 dia pós-natal do tipo selvagem neurônios simpáticos receberam microinjeções bovina do citocromo c (10 mg / mL), juntamente com rodamina-dextran para marcar células injetadas. Imagens mostram o mesmo campo de células a injeção imediatamente a seguir (0 h), ou nos horários indicados. Setas indicam células injetadas. Barra de escala, 20 mM. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Figura 2"/> Figura 2. XIAP deficiente neurônios são suscetíveis a microinjeção c citoplasmática citocromo. 5 º dia pós-natal neurônios simpáticos de camundongos knockout XIAP receberam microinjeções bovina do citocromo c (10 mg / mL), juntamente com rodamina-dextran para marcar células injetadas. Imagens mostram o mesmo campo de células a injeção imediatamente a seguir (0 h), ou 5 horas depois do citocromo c microinjeção (5 h). Barra de escala, 20 mM.

<ol>
	<li>Danial, N. N., Korsmeyer, 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=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116, 205-219 (2004).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+role+of+mitochondria+in+apoptosis.">The expanding role of mitochondria in apoptosis.</a> Genes Dev. 15, 2922-2933 (2001).</li><li>Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+apoptosis.">The biochemistry of apoptosis.</a> Nature. 407, 770-776 (2000).</li><li>Fuentes-Prior, P., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+structures+that+shape+caspase+activity,+specificity,+activation+and+inhibition.">The protein structures that shape caspase activity, specificity, activation and inhibition.</a> Biochem. J. 384, 201-232 (2004).</li><li>Tait, S. W., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+cell+death:+outer+membrane+permeabilization+and+beyond.">Mitochondria and cell death: outer membrane permeabilization and beyond.</a> Nat Rev Mol Cell Biol. 11, 621-632 (2010).</li><li>Brustugun, O. T., Fladmark, K. E., Doskeland, S. O., Orrenius, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induced+by+microinjection+of+cytochrome+c+is+caspase-dependent+and+is+inhibited+by+Bcl-2.">Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.</a> Cell Death Differ. 5, 660-668 (1998).</li><li>Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+induction+of+apoptosis+by+microinjection+of+cytochrome+c.+Bcl-XL+has+activity+independent+of+cytochrome+c+release.">Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-XL has activity independent of cytochrome c release.</a> J. Biol. Chem. 272, 30299-30305 (1997).</li><li>Deshmukh, M., Johnson, 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=Evidence+of+a+novel+event+during+neuronal+death:+development+of+competence-to-die+in+response+to+cytoplasmic+cytochrome+c.">Evidence of a novel event during neuronal death: development of competence-to-die in response to cytoplasmic cytochrome c.</a> Neuron. 21, 695-705 (1998).</li><li>Vaughn, A. E., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+inhibits+apoptosis+in+neurons+and+cancer+cells+by+redox+inactivation+of+cytochrome+c.">Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.</a> Nat. Cell Biol. 10, 1477-1483 (2008).</li><li>Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+apoptosis+by+Bcl-2:+release+of+cytochrome+c+from+mitochondria+blocked.">Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.</a> Science. 275, 1129-1132 (1997).</li><li>Gonzales, D. H., Neupert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+mitochondrial+c-type+cytochromes.">Biogenesis of mitochondrial c-type cytochromes.</a> J Bioenerg Biomembr. 22, 753-768 (1990).</li><li>Ellerby, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+cell-free+system+of+neuronal+apoptosis+-+comparison+of+premitochondrial,+mitochondrial,+and+postmitochondrial+phases.">Establishment of a cell-free system of neuronal apoptosis - comparison of premitochondrial, mitochondrial, and postmitochondrial phases.</a> J. Neurosci. 17, 6165-6178 (1997).</li><li>Neame, S. J., Rubin, L. L., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+cytochrome+c+activity+within+intact+neurons+inhibits+apoptosis.">Blocking cytochrome c activity within intact neurons inhibits apoptosis.</a> J. Cell Biol. 142, 1583-1593 (1998).</li><li>Potts, P. R., Singh, S., Knezek, M., Thompson, C. B., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+function+of+endogenous+XIAP+in+regulating+caspase+activation+during+sympathetic+neuronal+apoptosis.">Critical function of endogenous XIAP in regulating caspase activation during sympathetic neuronal apoptosis.</a> J. Cell Biol. 163, 789-799 (2003).</li><li>Estus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+gene+expression+in+neurons+during+programmed+cell+death:+identification+of+c-jun+as+necessary+for+neuronal+apoptosis.">Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.</a> J. Cell Biol. 127, 1717-1727 (1994).</li></ol>

Todos os procedimentos experimentais em animais foram aprovados pelo Institutional Animal Care e do Comitê Use na Universidade da Carolina do Norte.

A microinjeção de citocromo c diretamente no citoplasma das células é uma ferramenta original e poderosa que permite estudos de regulação pós-mitocondrial da apoptose. Importante, esta técnica permite a ativação direta da apoptose a jusante da mitocôndria sem o uso de agentes que causam dano celular ou mitocondrial. Embora este protocolo tem se concentrado em microinjeção de citocromo c para estudos sobre apoptose, os princípios gerais da microinjeção de pro...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> DM IRE2 microscópio invertido </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Puller Needle microinjeção </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO-202 micromanipulador </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet microinjetor </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> De parede fina de vidro Boroscilicate capilar com Microfilamentos </td><td> Sistemas de AM </td><td> 615000 </td><td> 4 Comprimento polegadas, 1,00 mm de diâmetro externo, 0,75 mm de diâmetro interno </td></tr><tr><td> Rodamina B isotiocianato-Dextran </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Peso molecular médio ~ 70000 Da </td></tr><tr><td> Citocromo c bovina Protein </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

Apoptose, ou morte celular programada, é uma via conservada e altamente regulado pelo qual as células morrem 1. Apoptose pode ser desencadeada quando as células encontrar uma ampla gama de tensões citotóxicos. Estes insultos iniciar cascatas de sinalização que acaba por causar a liberação de citocromo c do espaço intermembranar mitocondrial para o citoplasma 2. A liberação de citocromo c da mitocôndria é um evento-chave que desencadeia a rápida ativação das caspases, as proteases celular chave que acaba por executar a morte celular 3-4. A via de apoptose é regulada em pontos a montante ea jusante da liberação do citocromo c da mitocôndria 5. A fim de estudar a regulação pós-mitocondrial de ativação da caspase, muitos pesquisadores se voltaram para a microinjeção citoplasmática direta de holocytochrome c (heme-anexo) de proteína em células 6-9. Citocromo c é normalmente localizada na mitocôndria onde o apego de um grupo heme é necessária para habilitá-lo para ativar a apoptose 10-11. Portanto, para ativar diretamente caspases, é necessário para injetar a proteína c holocytochrome em vez do seu cDNA, porque enquanto a expressão de citocromo c de construções de cDNA resultará em alvo mitocondrial e apego heme, será seqüestrado de caspases citosólica. Assim, a microinjeção direta citosólica purificada heme ligado à proteína do citocromo c é uma ferramenta útil para imitar mitocondrial citocromo c release e apoptose sem o uso de insultos tóxicos que causam dano celular e mitocondrial. Neste artigo, descrevemos um método para a microinjeção de citocromo c de proteínas em células, usando fibroblastos de rato embrionárias (MEFs) e primário neurônios simpáticos como exemplos. Embora este protocolo incide sobre a injeção de citocromo c para as investigações da apoptose, as técnicas mostradas aqui também pode ser facilmente adaptado para microinjeção de outras proteínas de interesse.

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Neste protocolo, descrevemos a microinjeção direta citoplasmática de citocromo<em&gt; C</em&gt; Proteína em fibroblastos e primário neurônios simpáticos. Esta técnica permite a introdução do citocromo<em&gt; C</em&gt; Proteínas no citoplasma das células e imita a liberação de citocromo<em&gt; C</em&gt; A partir de mitocôndrias, que ocorre durante a apoptose.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K visualizações.  University of North Carolina. Neste protocolo, descrevemos a microinjeção direta citoplasmática de citocromo C Proteína em fibroblastos e primário neurônios simpáticos. Esta técnica permite a introdução do citocromo C Proteínas no citoplasma das células e imita a liberação de citocromo C A partir de mitocôndrias, que ocorre durante a apoptose.

Vídeo: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

Entrevista: apresentação de antígenos glicolipídeos por CD1d eo potencial terapêutico das células NKT Ativação

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Biologia

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Geração de transgênicos C. elegans Por biobalística Transformação

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análise de Cinética mRNA Nuclear Exportação em células de mamíferos por microinjeção

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualização da função respiratória mitocondrial usando Citocromo C Desidrogenase Oxidase / Succinato (COX / SDH) Histoquímica rotulagem dupla

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Síntese de um<em&gt; Em vivo</em&gt; Sonda Apoptosis MRI-detectável

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Molécula única imagem da regulação gênica<em&gt; Em vivo</em&gt; Uso de ativação Cotranslational por clivagem (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análise de apoptose em embriões Zebrafish por Whole-mount imunofluorescência para detectar Caspase 3 ativado

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

Estimulação da Citoplasmáticas DNA Sensing Pathways<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...