O original GFP-Pem1 foi um presente do Dr. Li Lei. Este trabalho foi financiado por concessões do NIH e da Ellison Medical Foundation para VG e AS

Neste protocolo, descrevemos o método para análise de DNA de reparo DSB com o repórter cromossomicamente integrada constrói 1,2 LAP onde são induzidos in vivo pela expressão transitória uma endonuclease corte rara I-SCEI 3. O ensaio integrado oferece a vantagem de analisar reparação DSB no contexto cromossômicas. No entanto, este protocolo requer célula passaging prolongada, que pode ser problemático quando se trabalha com pilhas. Uma abordagem alternativa é um ensaio extracromossómicos onde constrói repórter são digeridos in vitro com o I-SCEI ou endonucleases HindIII e então transfectado em células de DNA linear. O protocolo do ensaio exctrachromosomal 4 é semelhante ao ensaio descrito abaixo integrada com as seguintes modificações. Para o ensaio extracromossómicos omitir a integração de construções de repórter e, em vez co-transfecção as células com 0,5 mg de NHEJ plasmídeo repórter linearizada in vitro com o I-SCEI ou HindIII e 0,1 mcg de pDsRed2-N1 como controle de transfecção. Para o ensaio de RH co-transfecção duas repórter HR mg plasmídeo linearizado com I-SCEI ou HindIII e 0,1 mg pDsRed2-N1. Analisar as células por FACS três dias após a transfecção. O ensaio extracromossómicos permite a análise do DSB reparo em isolados primários, evitando extensa passaging celular e facilitando a análise de linhas de células múltiplas. 1. Constrói repórter NHEJ e HR A cassete repórter NHEJ 5 contém um gene GFP com uma engenharia 3 kb intron do gene Pem1 (GFP-Pem1). O intron Pem1 contém um exon adenoviral flanqueado por seqüências de reconhecimento de HindIII e I-SCEI endonucleases (Figura 1a) para a indução de LAP. Os sites de I-SCEI estão em posição invertida. I-SCEI tem uma seqüência de reconhecimento nonpalindromic, portanto, dois sites invertida gerar termina DNA incompatíveis (Figura 1c). DNA incompatíveis termina melhor imitar o LAP que ocorrem naturalmente. A cassete NHEJ intacta é GFP negativo como o exon adenoviral perturba o ORF GFP. Após a indução de LAP pelo I-SCEI, o exon adenoviral é removido e NHEJ restaura a função do gene GFP (Figura 2). A característica original deste repórter NHEJ é que ele pode detectar um amplo espectro de eventos NHEJ desde o intron pode tolerar exclusões e inserções. A cassete repórter HR 1 (Figura 1b) também é baseado no GFP-Pem1. Na cassete de RH, o primeiro exon do GFP-Pem1 contém uma deleção 22 bp combinado com a inserção de três sítios de restrição, I-SceI/HindIII/I-SceI. A supressão garante que GFP não pode ser reconstituído por um evento NHEJ. Os dois I-SCEI sites estão em posição invertida, de modo que I-SCEI deixa a digestão termina incompatíveis (Figura 1c). A primeira cópia de GFP-Pem1 é seguido por um exon promoter-less/ATG-less primeiro e intron de GFP-Pem1. A construção está intacta GFP-negativos. Após a indução de uma DSB pelo I-SCEI digestão do gene GFP funcional é reconstituído pela conversão intramolecular ou intermolecular gene entre as duas cópias mutantes do exon GFP-Pem1 primeiro. Desde a segunda cópia do gene GFP está faltando o códon ATG primeiro e segundo éxon, atravessando ou fita única de recozimento não irá restaurar a atividade GFP. Este projeto permite a detecção exclusiva de conversão de gene, que é o caminho HR predominantes em células de mamíferos (Figura 3). 2. Integração do Reporter Constrói no genoma <ol><li> Fazer uma preparação de alta qualidade de NHEJ ou repórter HR plasmídeo. Para melhores resultados use Qiagen Endo Kit grátis. Medir a concentração de plasmídeo e pureza, absorvência a 260/280 nm. </li><li> Linearizar 10 mg do plasmídeo repórter por digestão com 50 U de NheI em 50 ul (volume total da reação) por 6 horas em 37 ° C incubadora (não use o bloco seco, para evitar a evaporação). Purificar DNA a partir da solução digestão com Qiagen QiaexII Kit, DNA eluir em 20 l de 10 mM Tris-HCl pH 8.0. Medir a concentração de DNA e pureza, absorvência a 260/280 nm. Normalmente, você vai perder 20-30% de DNA durante a depuração. Confirme o rendimento ea digestão, executando uma pequena alíquota (2 mL) em um gel, junto com um repórter undigested construir. A construção repórter purificada linearizado pode ser armazenado a 20 ° C. </li><li> Em preparação para a transfecção, as células para trazer a melhor condição de crescimento. Se a partir de um frasco frozen, dividir as células duas vezes antes de transfecção. Se a partir de uma placa de células confluentes, dividi-las uma vez. </li><li> Crescer as células a confluência de 70-80% (geralmente dois dias, se banhado 5x10 mm Placa de 5 cells/100, por fibroblastos humanos normais). É fundamental para a melhor eficiência de transfecção para trazer as células em crescimento logarítmica. Cultura crescendo ativamente contém células em fase M (visto como células arredondadas ligado à superfície). </li><li> Transfect as células usando recomendações Amaxa fabricante seguintes Nucleofector é. Para fibroblastos uso NHDF solução e U20 programa. Para a transfecção, a colheita das células a partir de duas placas de 100 mm (~ 2x10 6 células). É fundamental não para mais de trypsinize as células. Parar tripsinização assim como 80% das células separado do prato. Ressuspender as células em solução NHDF. Células são sensíveis a solução NHDF, portanto, minimizar o tempo de incubação e misture delicadamente as células. Transfecção as células com 0,5 mg de construir repórter linearizada. Estas condições de transfecção, quando usado com normais resultado fibroblastos humanos na integração de uma única cópia de um repórter de construção para a maioria dos integrantes. </li><li> Selecione as células com construções repórter cromossomicamente integrado por adição de 1 mg / mL geneticin (G418) 24h após a transfecção. Continue seleção para dia 10/07. </li><li> Escolha as colônias G418 resistente (você pode escolher colônias individuais ou piscina clones resistentes). Expandir a cultura para cerca de cinco placas de 100 mm. Congelamento de três placas para uso futuro e ampliar outras duas placas com o número de células desejado (geralmente dez placas 100 mm são suficientes para cinco transfections). </li><li> Quando as células atingem confluência de 70-80% (dois dias após o plaqueamento 5x10 5 células por 100 mm para chapa de fibroblastos), as células estão prontas para serem transfectadas com I-SCEI expressar plasmídeo. </li></ol> 3. Indução de ORL por expressão transitória de I-SCEI endonuclease <ol><li> Transfectar ~ 2x10 6 células (duas placas) da cultura logaritmicamente em crescimento, com uma mistura de 5 g I-SCEI expressar plasmídeo e 0,1 mg pDsRed2-N1 (Clontech) como um controle de eficiência de transfecção usando Amaxa Nucleofector (use NHDF solução e U20 programa para fibroblastos ). </li><li> Ao mesmo tempo, preparar três controles de calibração para FACS por trasfecting ~ 2x10 6 células com (i) 5 mg EGFP-expressando plasmídeo, (ii) 5 mg pDsRed2-N1, (iii) 5 mg de um controle de plasmídeo que não expressa uma proteína fluorescente (estamos usando um plasmídeo HPRT minigene). </li><li> Cultivar as células por quatro dias para dar tempo suficiente para a expressão da GFP e proteínas DsRed. </li><li> (Opcional) Três dias após a transfecção verificar a eficiência da transfecção e expressão da GFP e proteínas fluorescentes DsRed por microscópio invertido com filtros para detecção de GFP e DsRed. </li></ol> 4. Análise da GFP + + e Células DsRed por citometria de fluxo <ol><li> Quatro dias após transfecção colheita I-SCEI células transfectadas e as células controle. Ressuspender as células em 500 mL de PBS e transferir células para os tubos FACS. Nesta fase é fundamental para colher todas as células e têm células de forma arredondada. Para conseguir isso, é recomendado o uso de um maior tempo de tripsinização ou uma forte tripsina. Confirmar que 99% das células são separadas do prato e ter forma redonda, observando as células ao microscópio. Manter as células no gelo protegido da luz (tampa balde de gelo com papel alumínio). É possível armazenar as células no gelo por algumas horas. Para obter os melhores resultados analisar as células imediatamente após a colheita. </li><li> Calibrar FACS com GFP, DsRed, e os controles negativos. Ajuste de tensão e compensação de cor de modo a incluir todas as células fluorescentes na análise (Figura 4). Tenha em mente que a intensidade de fluorescência das amostras experimental será menor que a dos controles. </li><li> I-analisar SCEI transfectadas células por FACS. Contamos pelo menos 20.000 células para cada tratamento. Para evitar a interferência potencial entre GFP e de fluorescência DsRed é recomendado para manter o percentual de GFP + ou + DsRed células abaixo de 25%. Se o percentual de + DsRed ou células GFP + é maior reduzir a quantidade de pDsRed2-N1 ou I-SCEI plasmídeo. Em nossos experimentos que só precisava ajustar a quantidade de pDsRed2-N1 do plasmídeo, mas não do plasmídeo I-SCEI. </li><li> A porcentagem de células GFP + corresponde à eficiência do reparo de DNA DSB eo percentual de células + DsRed indica a eficiência de transfecção. Calcular a eficiência relativa de reparo de DNA DSB como a razão de células GFP + + para as células DsRed. </li><li> As junções final reparado pode ser seqüenciadas para testar a precisão de reparação. O repórter constrói conter E.coli origem de replicação e genes de resistência a canamicina (Figura 1). Isto permite resgatar as construções através da digestão do DNA genômico com enzima EcoRI, recircularization e transformação em células competentes E. coli. Os plasmídeos resgatados são então analisados ​​por digestão de restrição e seqüenciamento. </li></ol> 5. Resultados representante Resultados típicos da FACS transfections e controle de NHEJ e experiências de RH são mostrados nas Figuras 4 e 5. Intensidade de fluorescência e do número de células fluorescentes será menor nas células transfectadas I-SCEI contendo integrated NHEJ e constrói HR (Figura 5) em comparação com os controles (Figura 4), devido à diferença no número de cópias de GFP e constrói DsRed nestas células. Cada célula no experimento contém apenas uma cópia do constructo repórter integrado, eventos de reparo, portanto, bem sucedido vai reconstituir o gene GFP em uma fração das células. Transfecção com 0,1 mg pDsRed2-N1 em fibroblastos humanos oferece cerca de 1 cópia do plasmídeo por célula. A eficiência do NHEJ e HR é calculado como uma proporção de GFP + / + DsRed células. NHEJ é um processo mais eficiente que o HR em células humanas 2. Em fibroblastos humanos a eficiência NHEJ é tipicamente 0,6-1,3, e eficiência de RH é 0,05-0,3. Desde eficiência DSB é medido como uma relação de GFP + / + DsRed células variações na mistura de I-SCEI e DsRed2-N1 plasmídeos podem afetar o resultado. A qualidade plasmídeo também podem afetar os resultados, alterando a eficiência de transfecção. Portanto, para obter medidas consistentes, é importante usar a mesma mistura plasmídeo dentro de um experimento. Além disso, a eficiência de reparo DSB é afetada pelo tipo de célula, a fase do ciclo celular, e da localização cromossômica das construções integradas. <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Figura 1" /> Figura 1. Reporter construções para a análise de DNA de reparação por DSB NHEJ (A) e HR (B). (C) DNA incompatíveis termina gerada por digestão de dois invertido I-SCEI locais SD, splice doadores; SA, splice aceitador; praças, locais de poliadenilação; Neo / Kana, única ORF controlado por dois promotores:. SV40 resistência a neomicina conferir em mamíferos células, e β-lactamase resistência kanamicyn conferir em E. coli; Ori, E. Origem coli pUC de replicação. <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Figura 2" /> Figura 2. Reporter construir para a análise de NHEJ Neste construir o gene GFP está inativo;. Porém após a indução de um NHEJ DSB e bem sucedida a construção torna-se GFP +. Mostrado abaixo é um produto de reparação de este repórter por NHEJ. <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Figura 3" /> Figura 3. Reporter construir para a análise de RH pela conversão gene. Upon indução da LAP pelo I-SCEI, HR pelo gene de conversão (GC) reconstitui um gene GFP ativo. Mostrados abaixo são produtos de reparação deste repórter pelas vias de reparo de DNA: GC, crossing-over (X-over), único fio de recozimento (SSA), e NHEJ. X-over produto de reparação é mostrado para recombinação intramolecular, recombinação exramolecular vai dar o mesmo produto, mas localizado em um cromossomo. Genes que expressam a proteína GFP ativa (GFP +) são indicados pela cor verde. <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Figura 4" /> Figura 4. Calibração dos parâmetros para análise FACS. (A) Os fibroblastos transfectados com o plasmídeo pHPRT, usado como controle negativo para excluir as células auto fluorescente. (B) fibroblastos transfectados com pEGFP plasmídeo, usado para definir a propagação de células GFP + (área verde). (C) Os fibroblastos transfectados com pDsRed2-N1 do plasmídeo, usado para definir a propagação de células + DsRed (área vermelha). <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Figura 5" /> Figura 5. Resultados típicos da análise de NHEJ (A) e HR (B) em fibroblastos humanos normais com construções repórter cromossomicamente integrado.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

Não há conflitos de interesse declarados.

O NHEJ fluorescentes e ensaios repórter HR fornecem uma maneira quantitativos para medir separadamente cada via de reparo DSB in vivo. Os ensaios são muito sensíveis, como FACS pode detectar 10 células GFP + de 20.000 células. Os ensaios podem ser adaptados para medir eventos de reparo em &quot;tempo real&quot; através da detecção do aparecimento de células GFP + em minutos ou horas após a indução de LAP 2. Além disso, a análise de células GFP + não depende de proliferação celular a...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
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analysis of dna double-strand break (dsb) repair in mammalian cells

DNA dupla fita-breaks são as lesões mais perigosas DNA que podem levar à perda maciça de informações genéticas e morte celular. Células LAP reparar usando duas vias principais: nonhomologous final juntar (NHEJ) e recombinação homóloga (HR). Perturbações de NHEJ e HR são frequentemente associados com o envelhecimento prematuro e tumorigênese, por isso é importante ter uma forma quantitativa de medir cada via de reparo DSB. Nosso laboratório desenvolveu constrói repórter fluorescentes que permitem a medição sensível e quantitativa de NHEJ e RH. As construções são baseadas em um gene GFP engenharia contendo sítios de reconhecimento para uma rara corte endonuclease I-SCEI para a indução de LAP. As construções de partida são GFP negativo como o gene GFP é inativado por um exon adicional, ou por mutações. Reparo com sucesso dos intervalos I-SCEI induzida por NHEJ ou HR restaura o gene GFP funcional. O número de células GFP positivas contadas por citometria de fluxo fornece uma medida quantitativa de eficiência ou NHEJ HR.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Este artigo descreve GFP baseada em fluorescência<em&gt; In vivo</em&gt; Ensaios que quantificar separadamente recombinação homóloga e nonhomologous final juntar em células de mamíferos.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K visualizações.  University of Rochester. Este artigo descreve GFP baseada em fluorescência In vivo Ensaios que quantificar separadamente recombinação homóloga e nonhomologous final juntar em células de mamíferos.

Vídeo: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfecção de DNA de mamíferos usando músculos esqueléticos In Vivo Eletroporação

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Biologia

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Intranucleares microinjeção de DNA em Dissociated neurônios de mamíferos adultos

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulso-chase Análise de Cadeias N-linked Açúcar de Glicoproteínas em células de mamíferos

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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 ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.