Name: culture and transfection of zebrafish primary cells
Uploaded: 2018-08-17T13:00:00
Description: Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Agradecemos a T. Fritsch, A. lobo-Asseburg, I. Linde e S.-M. Tokarski para excelentes cuidados com animais e suporte técnico. Agradecemos a todos os membros do laboratório Köster discussões intensas e útil. Reconhecemos com gratidão financiamento pela Deutsche Forschungsgemeinschaft (KO 1949/5-1) e o Estado Federal da Baixa Saxónia, Niedersächsisches Vorab (VWZN2889).

Todo trabalho animal aqui descrito está em conformidade com regulamentos legais (EU-Directiva 2010/63). Manutenção e manipulação de peixes foi aprovado pelas autoridades locais e o bem-estar dos animal representativo da Universidade Técnica de Braunschweig e Baixa Saxônia estado escritório de defesa do consumidor e da segurança alimentar (elevam, Oldenburg, Alemanha; AZ. parágrafo 4º (02.05) TSchB TU BS).
1. preparação da escola primária de células de embriões de peixe-zebra
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+chromosomal+localizations+of+two+human+genes+encoding+synaptobrevins+1+and+2.">Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2.</a> Journal of Biological Chemistry. 265, 17267-17273 (1990).</li><li>Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+the+environmental+sensitivity+of+yellow+fluorescent+protein.+Mechanism+and+applications.">Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.</a> Journal of Biological Chemistry. 276, 29188-29194 (2001).</li><li>Shaner, N. 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Aqui, apresentamos dois protocolos diferentes de cultura primária de células de 2 embriões de zebrafish dpf ou cérebro adulto zebrafish.
A preparação das culturas de célula primária de 2 dpf zebrafish é relativamente fácil de executar para qualquer pessoa com experiência em técnicas de cultura de célula básica. No entanto, para obter resultados reprodutíveis e boas, um número suficiente de embriões como material de partida é crucial (100 é o mínimo). Durante o levantamento ...

O peixe-zebra (Danio rerio, d. rerio) é um modelo popular vertebrado para inúmeros campos de pesquisa básica e biomédico1. Zebrafish embriões se desenvolvem rapidamente ex utero, são transparentes e ajuste sob um microscópio, proporcionando excelentes pré-requisitos para estudar o desenvolvimento de vertebrados em um organismo vivo. Devido a rastreabilidade genética de zebrafish2, foram criadas muitas linhas de repórter transgénicos estável com expressão de tipo específico de célula de vários marcadores fluorescentes permitindo a observação de populações de células específicas. A comunidade de zebrafish oferece uma ampla variedade de chamados linhas Gal4-motorista que carregam um transgene expressando o Kal4TA4 sintético (ou o equivalente de KalTA3-GalFF) gene com o domínio de Gal4-DNA-ligando de levedura fundido a ativação transcricional viral domínios sob o controle dos realçadores de tipo específico de célula. Estas linhas de motorista se cruzam linhas de efetor que carregam transgenes consistindo de uma definido upstream ativando sequência (UAS) fundida a um gene repórter. A proteína Kal4TA4 vincula-se ao elemento UAS, ativando assim a expressão seletiva-tipo de célula do gene repórter3,4. Esta abordagem permite estudos combinatórios altamente diversificados de quase todos os elementos disponíveis enhancer e repórter em animais transgénicos-duplo.
No entanto, imagens ao vivo em profundidade com foco em células individuais ou seus conteúdos subcellular é limitada em um embrião inteiro e constante mudança. Para abordar questões biológicas de célula específica com maior resolução, o uso de culturas de células, muitas vezes é preferível. Existem algumas linhas de célula de zebrafish, mas eles são considerados como altamente selecionado5,6,7 e sua propagação geralmente é demorada. Além disso, todas as linhas de celular disponíveis são fibroblastos derivados, limitando os experimentos utilizando cultura de células de um tipo de células. Portanto, estabelecemos um protocolo eficiente e fácil de usar para preparar as células primárias diretamente do zebrafish embriões e cérebro adulto zebrafish, juntamente com abordagens para aumentar a longevidade da cultura e ampliar a diversidade de cultivo tipos de células. Além disso, apresentamos um procedimento para transfect embrionário as células primárias com construções de expressão para marcadores fluorescentes organela. Assim, estruturas subcelulares e morfologias celulares podem ser analisadas com alta resolução espacial e temporal em tipos de células distintas que retêm suas principais características.

<table>
	<tbody>
		<tr>
			<td>Fish lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AB (wild-type)</td>
			<td></td>
			<td></td>
			<td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td>
		</tr>
		<tr>
			<td>Tg(ptf1a:eGFP)jh1</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007)</td>
		</tr>
		<tr>
			<td>Tg(XITubb:DsRed)zf148</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&#160; (Peri and N&#252;sslein-Volhard, 2008)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>model 5804 R</td>
		</tr>
		<tr>
			<td>ChemiDoc MP imaging system</td>
			<td>BioRad</td>
			<td></td>
			<td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td>
		</tr>
		<tr>
			<td>confocal laser scanning microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model SP8, equipped with 28 &#176;C temperature box and a 63 x objective</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model DM5500B, equipped with 28 &#176;C temperature box and a 40 x objective</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell with capacitance extender</td>
			<td>BioRad</td>
			<td>1652661</td>
			<td>electroporation device</td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>GFL</td>
			<td></td>
			<td>model 3011</td>
		</tr>
		<tr>
			<td>incubator for cell culture (28 &#176;C)</td>
			<td>Memmert</td>
			<td></td>
			<td>model incubator I</td>
		</tr>
		<tr>
			<td>incubator for embryos (28 &#176;C)</td>
			<td>Heraeus</td>
			<td></td>
			<td>type B6120</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>model TELAVAL 31</td>
		</tr>
		<tr>
			<td>micro pipettes</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sterile work bench</td>
			<td>Bio Base</td>
			<td></td>
			<td>with laminar flow and UV light</td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td>Style 5, Inox</td>
		</tr>
		<tr>
			<td>vertical tube rotator</td>
			<td>Labinco B.V.</td>
			<td></td>
			<td>model LD-79</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab Software</td>
			<td>BioRad</td>
			<td></td>
			<td>for the ChemiDoc MP imaging system from BioRad</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>for both confocal and epifluorescent microscopes from Leica Microsystems</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCS-DCX-tdTomato</td>
			<td>K&#246;ster Lab</td>
			<td># 1599</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-eGFP</td>
			<td>K&#246;ster Lab</td>
			<td># 7</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-H2B-mseCFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2379</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-mClover</td>
			<td>K&#246;ster Lab</td>
			<td># 3865</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-MitoTag-YFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2199</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-ss-RFP-KDEL</td>
			<td>K&#246;ster Lab</td>
			<td># 4330</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-VAMP1-mCitrine</td>
			<td>K&#246;ster Lab</td>
			<td># 2291</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pSK-UAS:mCherry</td>
			<td>K&#246;ster Lab</td>
			<td># 1062</td>
			<td>based on the pBluescript-backbone of Stratagene</td>
		</tr>
		<tr>
			<td>Plasmid numbers refer to the database entries of the K&#246;ster lab. Plasmids are available upon request.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plastic and glass ware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Cell Strainer (40 &#181;m)</td>
			<td>FALCON</td>
			<td>REF 352340</td>
			<td>distributed by BD Bioscience, used as &#8220;landing net&#8221; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td>
		</tr>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falconic tube</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>83.3924.005</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyStrainer (40 &#181;m)</td>
			<td>Greiner Bio-One</td>
			<td>542 040</td>
			<td>with venting slots; used to filter cells after collagenase-mediated dissociation</td>
		</tr>
		<tr>
			<td>electroporation cuvette (0.4 cm)</td>
			<td>Kisker</td>
			<td>4905022</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslips</td>
			<td>Heinz Herenz Medizinalbedarf GmbH</td>
			<td>1051201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher Scientific (Menzel Gl&#228;ser)</td>
			<td>631-0845</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Henneberg-Sander GmbH</td>
			<td>9020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (plastic; 3 mL)</td>
			<td>A. Hartenstein</td>
			<td>PP05</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>821473</td>
			<td>for zebrafish embryos</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>Sarstedt</td>
			<td>Blue (1000 &#181;l): 70762; Yellow (200 &#181;l): 70760002; White (10 &#181;L): 701116</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 3 cm)</td>
			<td>TPP Techno Plastic Products AG</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 6 cm)</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td>for brain dissection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Roth</td>
			<td>0601.1</td>
			<td></td>
		</tr>
		<tr>
			<td>4 % paraformaldehyde in 1 x PBS</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium nitrate tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol p.a. 100%</td>
			<td>Sigma-Aldrich</td>
			<td>46139</td>
			<td></td>
		</tr>
		<tr>
			<td>goat &#945;-mouse IgG (Fc specific) FITC conjugated</td>
			<td>Thermo Fisher Scientific</td>
			<td>31547</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>high vacuum grease</td>
			<td>DOW CORNING</td>
			<td>3826-50</td>
			<td>silicon grease used for self-made glass bottom dishes</td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Merck</td>
			<td>105886</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Serva</td>
			<td>29198.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Tubulin, Acetylated antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua-Poly/Mount (mounting medium)</td>
			<td>Polyscience</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Biochrom</td>
			<td>L 7240</td>
			<td></td>
		</tr>
		<tr>
			<td>potasssion chloride</td>
			<td>Merck</td>
			<td>104938</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Roth</td>
			<td>68514-61-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-X Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioRad</td>
			<td>1610407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco by Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase (Type 2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17101015</td>
			<td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pronase (from Streptomyces griseus)</td>
			<td>Roche</td>
			<td>11459643001</td>
			<td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Medium and solutions for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 x PBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-169</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>CO2-independent medium</td>
			<td>Gibco by Life Technologies</td>
			<td>18045054</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>filtrated bovine serum (FBS)</td>
			<td>PAN-Biotech</td>
			<td></td>
			<td>individual batch</td>
		</tr>
		<tr>
			<td>glutamine 100 x</td>
			<td>Gibco by Life Technologies</td>
			<td>25030081</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium</td>
			<td>Gibco by Life Technologies</td>
			<td>11415049</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>PenStrep (10,000 units/mL)</td>
			<td>Gibco by Life Technologies</td>
			<td>15140148</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
	</tbody>
</table>

culture and transfection of zebrafish primary cells

Zebrafish embriões são transparentes e desenvolvem rapidamente fora da mãe, permitindo assim excelente na vivo por imagens do processos biológicos dinâmicos em um vertebrado intacto e em desenvolvimento. No entanto, a imagem detalhada das morfologias de tipos distintos de células e estruturas subcelulares é limitada em montagens de toda. Portanto, nós estabelecemos um protocolo eficiente e fácil de usar para as células primárias ao vivo de cultura de embriões de peixe-zebra e tecido adulto.
Em breve, 2 dpf zebrafish embriões são dechorionated, deyolked, esterilizados e dissociado de células únicas com colagenase. Após uma etapa de filtração, as células primárias são banhadas em pratos de vidro inferior e cultivadas por vários dias. Culturas frescas, diferenciada de termo tanto quanto tempo os, podem ser usadas para estudos de imagiologia confocal de alta resolução. A cultura contém diferentes tipos de células, com miócitos estriados e neurônios sendo proeminente no revestimento de poli-L-lisina. Para especificamente rótulo subcellular estruturas por proteínas marcador fluorescente, também estabelecemos um protocolo de eletroporação que permite o transfeccao de Plasmideo DNA em diferentes tipos de células, incluindo neurônios. Assim, na presença do operador definido de estímulos, comportamento complexo celular e dinâmica intracelular de células primárias zebrafish pode ser avaliada com alta resolução espacial e temporal. Além disso, usando o cérebro adulto zebrafish, demonstramos que a técnica de dissociação descrita, bem como as condições básicas de cultivo, também trabalham para tecido adulto zebrafish.

A Figura 1 mostra uma imagem de luz transmitida de uma cultura típica derivada de embriões de tipo selvagem com miócitos estriados e aglomerados de células neurônio-como sendo o mais abundante. Para identificar determinados tipos de células mais facilmente, uma linha de transgênica com expressão de tipo específico de célula de uma proteína fluorescente pode ser usado (Figura 1 H).
<p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

Apresentamos um protocolo eficiente e fácil de usar para a preparação de culturas de células primárias de zebrafish embriões para transfeccao e imagens de células vivas, bem como um protocolo para preparar as células primárias do cérebro adulto zebrafish.

Cultura e transfecção de células primárias de Zebrafish

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