Name: culture and transfection of zebrafish primary cells
Uploaded: 2018-08-17T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

T. Fritsch, A. kurt-Asseburg, ı. Linde ve S.-M teşekkür ederiz. Tokarski mükemmel hayvan bakım ve teknik destek için. Yoğun ve yararlı tartışmalar için Köster laboratuvar Tüm üyeler için minnettarız. Deutsche Forschungsgemeinschaft (KO 1949/5-1) ve Federal Devlet Aşağı Saksonya, Niedersächsisches Vorab (VWZN2889) tarafından finansman minnetle anıyoruz.

<ol>
	<li>Ablain, J., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+fish+and+men:+using+zebrafish+to+fight+human+diseases.">Of fish and men: using zebrafish to fight human diseases.</a> Trends in Cell Biology. 23, 584-586 (2013).</li><li>Sassen, W. A., K&#246;ster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+toolbox+for+genetic+manipulation+of+zebrafish.">A molecular toolbox for genetic manipulation of zebrafish.</a> Advances in Genomics and Genetics. , 151 (2015).</li><li>Scheer, N., Campos-Ortega, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Gal4-UAS+technique+for+targeted+gene+expression+in+the+zebrafish.">Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.</a> Mechanisms of Development. 80, 153-158 (1999).</li><li>K&#246;ster, R. W., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+transgene+expression+in+living+zebrafish+embryos.">Tracing transgene expression in living zebrafish embryos.</a> Developmental Biology. 233, 329-346 (2001).</li><li>Driever, W., Rangini, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+cell+line+derived+from+zebrafish+(Brachydanio+rerio)+embryos.">Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 29A, 749-754 (1993).</li><li>Badakov, R., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+primary+zebrafish+fibroblasts+by+nucleofection.">Efficient transfection of primary zebrafish fibroblasts by nucleofection.</a> Cytotechnology. 51, 105-110 (2006).</li><li>Senghaas, N., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+transfecting+zebrafish+PAC2+fibroblast+cells.">Culturing and transfecting zebrafish PAC2 fibroblast cells.</a> Cold Spring Harbor Protocols. , (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Basic methods in cellular and molecular biology. Using a hemacytometer to count cells. Journal of Visualized Experiments Available from: <a target="_blank" href="https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells">https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells</a> (2017)</li><li>Rupp, R. A., Snider, L., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+embryos+regulate+the+nuclear+localization+of+XMyoD.">Xenopus embryos regulate the nuclear localization of XMyoD.</a> Genes &amp; Development. 8, 1311-1323 (1994).</li><li>Piperno, G., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+specific+for+an+acetylated+form+of+alpha-tubulin+recognize+the+antigen+in+cilia+and+flagella+from+a+variety+of+organisms.">Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.</a> Journal of Cell Biology. 101 (6), 2085-2094 (1985).</li><li>Barden, J. A., Miki, M., Hambly, B. D., Dos Remedios, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+phalloidin+and+nucleotide-binding+sites+on+actin.">Localization of the phalloidin and nucleotide-binding sites on actin.</a> European Journal of Biochemistry. 162 (3), 583-588 (1987).</li><li>Kapuscinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAPI:+a+DNA-specific+fluorescent+probe.">DAPI: a DNA-specific fluorescent probe.</a> Biotechnic &amp; Histochemistry. 70 (5), 220-233 (1995).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, E1717 (2010).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Stornaiuolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KDEL+and+KKXX+retrieval+signals+appended+to+the+same+reporter+protein+determine+different+trafficking+between+endoplasmic+reticulum,+intermediate+compartment,+and+Golgi+complex.">KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex.</a> Molecular Biology of the Cell. 14, 889-902 (2003).</li><li>Lithgow, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+proteins+to+mitochondria.">Targeting of proteins to mitochondria.</a> FEBS Letters. 476, 22-26 (2000).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nature Biotechnology. 20, 87-90 (2002).</li><li>Sassen, W. A., Lehne, F., Russo, G., Wargenau, S., D&#252;bel, S., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+zebrafish+primary+cell+culture+for+transfection+and+live+cellular+and+subcellular+imaging.">Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.</a> Developmental Biology. 430, 18-31 (2017).</li><li>Horesh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doublecortin,+a+stabilizer+of+microtubules.">Doublecortin, a stabilizer of microtubules.</a> Human Molecular Genetics. 8, 1599-1610 (1999).</li><li>Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22, 1567-1572 (2004).</li><li>Distel, M., Hocking, J. C., Volkmann, K., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+neither+persistently+leads+migration+nor+determines+the+site+of+axonogenesis+in+migrating+neurons+in+vivo.">The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo.</a> Journal of Cell Biology. 191, 875-890 (2010).</li><li>Matsuda, T., Miyawaki, A., Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+dynamics+inside+cells+using+a+rationally+designed+photoconvertible+protein.">Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein.</a> Nature Methods. 5, 339-345 (2008).</li><li>Archer, B. T., Oz&#231;elik, T., Jahn, R., Francke, U., S&#252;dhof, T. 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. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+monomeric+green+fluorescent+protein+derived+from+Branchiostoma+lanceolatum.">A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.</a> Nature Methods. 10, 407-409 (2013).</li><li>Campbell, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomeric+red+fluorescent+protein.">A monomeric red fluorescent protein.</a> Procedings of the National Academy of Sciences of the United States of America. 99, 7877-7882 (2002).</li><li>Peri, F., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuronal+degradation+by+microglia+reveals+a+role+for+v0-ATPase+a1+in+phagosomal+fusion+in+vivo.">Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.</a> Cell. 133, 916-927 (2008).</li><li>Godinho, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. 132, 5069-5079 (2005).</li><li>Jusuf, P. R., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ptf1a+is+expressed+transiently+in+all+types+of+amacrine+cells+in+the+embryonic+zebrafish+retina.">Ptf1a is expressed transiently in all types of amacrine cells in the embryonic zebrafish retina.</a> Neural Development. 4, 34 (2009).</li><li>Kani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proneural+gene-linked+neurogenesis+in+zebrafish+cerebellum.">Proneural gene-linked neurogenesis in zebrafish cerebellum.</a> Developmental Biology. 343, 1-17 (2010).</li><li>Distel, M., Wullimann, M. F., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Gal4+genetics+for+permanent+gene+expression+mapping+in+zebrafish.">Optimized Gal4 genetics for permanent gene expression mapping in zebrafish.</a> Procedings of the National Academy of Sciences of the United States of America. 106, 13365-13370 (2009).</li><li>Choorapoikayil, S., Overvoorde, J., den Hertog, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+cell+lines+from+zebrafish+embryos+and+tumors.">Deriving cell lines from zebrafish embryos and tumors.</a> Zebrafish. 10, 316-332 (2013).</li></ol>

Yazarlar ifşa gerek yok.

Burada, iki farklı protokol 2 dpf zebra balığı embriyo veya yetişkin zebra balığı beyin Kültür primer hücre için mevcut.
2 dpf zebra balığı birincil hücre kültürleri hazırlanması temel hücre kültürü teknikleri deneyimi olan herkes için gerçekleştirmek nispeten kolaydır. Ancak, iyi ve tekrarlanabilir sonuçlar elde etmek için yeterli sayıda embriyo malzeme başlangıç olarak çok önemlidir (100 olduğunu en azından). Embriyo yükseltme sırasında tüm olası kaynaklarını kirlenme mikroplar ve parazitler miktarını mümkün olduğunca düşük tutmak için kaçınılmalıdır. Kuluçka embriyoların etanol içinde en kritik — bu adım ayrıntılı Sterilizasyon olun ama etanol hücre ve doku zarar organik bir çözücü olduğu için pek de uzun kadar uzun olması gerekiyor.
Zamanlama sırasında beyin doku veya 2 dpf zebra balığı embriyo ayrılma enzimatik esastır. Kuluçka süresi aşılmaması ve tek hücreli aşamaya tam ayrılma başarılı bir kez enzim içeren orta hızlı bir şekilde çıkarmak. Yine de, tip 2 collagenase hücre zarı zarar vermez ve değil güçlü olarak FBS, tripsin33olarak tarafından engellenir. Bu özellikler sayesinde, biz daha yüksek verim ve hassas hücre tiplerinin canlılığı ile sonuçlanan FBS huzurunda ayrılma hücre canlılığı desteklemek ve hücre hasarı, azaltılması önemli bir ek olarak gerçekleştirmek başardık.
Temsilcisi sonuçlarımız tarafından gösterildiği gibi farklı hücre tipleri de morfoloji veya hücre türüne özgü transgenes floresan muhabirleri kodlama ifade tarafından tanımlanan olabilir. İkinci canlı hücre görüntüleme belirli hücre nüfus19gerçekleştirirken çok uygundur. Ancak, tutarlı sonuçlar sağlamak için floresans mikroskobu tarafından 2 dpf embriyo ilgili artırıcı ifade desenini doğrulamak için önerilir.
Embriyonik zebra balığı primer hücre hücre altı yapılarını görüntüleme için izin vermek için bir adım Protokolü floresan organel işaretleri bir seçim için kodlama plazmid DNA transfect kurduk. Bu yöntemle transfected hücre oranı nispeten düşük ama güvenilir ve yeterli hücre sayıları19görüntüleme canlı hücre için sağlar. Başarılı bir transfection için süspansiyon hücre sayısı doğru belirlenmesi önemli bir adımdır. Ortalama içinde 106 hücre yaklaşık 90 2 dpf embriyo elde edilebilir, ancak doğru hücre önceki adım sayma zorunlu19. Ancak, Cos-7 veya HeLa gibi ortak memeli hücre hatları ile karşılaştırıldığında, birincil hücrelerden 2 dpf zebra balığı embriyo boyutunda çok çeşitlidir, ama ortalama (özellikle nöronlar) oldukça küçük, hangi Neubauer odasında nispeten zor sayım yapabilirsiniz. Aynı sonraki görüntüleme uygulamaları için geçerlidir. Tatmin edici bu küçük zebra balığı hücrelerdeki hücre altı yapıları görselleştirmek için yüksek çözünürlüklü bir Gelişmiş floresans mikroskobu tercihen mikroskobu tarama confocal lazer gereklidir.
Eğitimi gerektirdiğinden primer hücre--dan yetişkin zebra balığı beyin hazırlanması daha gelişmiş hayvan anatomi ve beyin dokusu14ayıklanıyor. Buna ek olarak, Steril koşullar zaten ayıklanan beyin kirletmek değil steril tezgah dışında muhafaza edilmesi gerekir. Ancak, sonraki adımları kaplama ve ayrılma ile ilgili ilk protokole karşılaştırılabilir ve diğer dokulara kas ya da karaciğer gibi yetişkin balık da uygulanabilir.
Her iki iletişim kuralları için ana hücre tipleri kültür, nöronlar gibi yetiştirmek zor sınırlı canlılığı kısıtlamadır. 3 standart koşullar kullanarak dap sonra embriyonik zebra balığı primer hücre yoğunluğu şiddetle böylece deneyler19görüntüleme için zaman aralığı sınırlama azalır. Çoğunlukla fiboblast benzeri hücreler özel olarak uyarlanmış expedients yokluğunda hayatta.
Karmaşıklığı hücre tiplerinin kültüründe survival oranı artırmak için başarılı bir şekilde iyi'nın L-15 orta test ettik. Daha fazla nöron teşvik katkı B-27 nöronal belirli kültür için ile desteklenmiştir. Nöronal hücre hücre kültüründe ayırt etmek ve birkaç gün için hayatta kalmak için gözlemlenmiştir ne zaman tüm embriyo elde edilen (Şekil 2F-ben) yanı sıra--dan yetişkin beyin gibi (Şekil 4E). Buna ek olarak, primer hücre yüksek yoğunlukları (Örneğin, 0,25 x 10 100 mm2başına6 hücre), kaplama düşük yoğunlukları (G. Russo, ilk sonuçlar) aksine hücre canlılığı artırır.
Özel kültür medya ve takviyeleri kullanımı, biz nasıl farklı yüzeylerde kullanımı son kültürü oluşturmaktadır ve seçime bağlı parametre olarak bir alt kümesini hücre büyümesini teşvik kullanılan hücre tipleri çeşitli üzerinde doğrudan bir etkisi vardır rapor türleri. Güçlü fibroblast benzeri hücre adezyon ve nükleer silahların yayılmasına karşı (Şekil 1E, F) kolaylaştırmak için doku kültürü tedavi plastik poli-L-lizin kaplama için kıyasla görünüyor. Aynı zamanda hücreleri bağlı çok sayıda hücre türleri destekler ve belirli adezyon molekülleri (gibi nöronlar) düzgün bağlı değil, büyümek, ya da üzerinde işlem görmüş plastik ayırt etmek ama özel yapıştırıcı destek tercih ederim. Bu yüzden öneririz, ne zaman bir fibroblast veya epitel-benzeri kültür33, hücre kültürü yemekleri kaplama için poli-L-lizin veya diğer hücre özel yüzeylerde kullanmak için almak üzere tasarlanmamıştır.
Daha fazla olabilir zebra balığı in vivo çalışmalar tamamlayacak bir geliştirilmiş ve belirli hücre tipleri ekimi için uyarlanmış ve birçok çeşitli uygulama için kullanılan gibi bizim Protokolü birincil hücre kültürü kullanımı için bir başlangıç noktası göz önünde bulundurun.

Zebra balığı (Danio rerio, D. rerio) temel ve Biyomedikal araştırma1çok sayıda alanlar için omurgalı popüler bir modeldir. Zebra balığı embriyolar rahimhızla geliştirmek, şeffaf ve böylece canlı bir organizma olarak omurgalı geliştirme eğitim mükemmel önkoşulları sağlayan bir mikroskop altında Sığdır. Zebra balığı2genetik tractability nedeniyle, birçok istikrarlı transgenik muhabir satırları hücre türüne özgü ifade çeşitli floresan işaretleri ile belirli hücre popülasyonlarının gözlem için izin kurulmuştur. Zebra balığı topluluk sentetik Kal4TA4 (veya KalTA3-eşdeğer GalFF) ifade bir transgene taşımaya sözde Gal4-sürücü satırları geniş sunar gen Gal4 DNA'ya bağlanıcı etki alanı Maya ile erimiş viral transkripsiyon harekete geçirmek hücre türüne özgü arttırıcılar kontrolü altındaki etki alanları. Bu sürücü satırları transgenes bir muhabir gen erimiş bir tanımlanmış ters yönde harekete geçirmek sırasında (UAS) oluşan taşımak efektör satırlar için üzerinden geçilen. Kal4TA4 protein böylece hücre türü Seçici ifade muhabir gen3,4aktive UAS öğesine bağlar. Hemen hemen tüm kullanılabilir artırıcı ve muhabir'ndaki öğelerin çift transjenik hayvanlar çok çeşitli Kombinatorik çalışmaları için bu yaklaşım sağlar.
Ancak, tek tek hücreler veya hücre altı içeriklerini odaklanmak ile derinlemesine canlı görüntüleme bir bütün ve sürekli değişen embriyo sınırlıdır. Belirli hücre biyolojik soruları ile en yüksek çözünürlük için hücre kültürlerinde kullanımı genellikle tercih edilir. Zebra balığı, bazı hücre satır yok, ama ağırlıklı olarak seçilen5,6,7 olarak kabul edilir ve onların yayılma kez zaman alır. Ayrıca, tüm kullanılabilir hücre satırlarını türetilmiş, fibroblast hücreleri bir tür hücre kültürü kullanarak deneyler sınırlama vardır. Bu nedenle, biz doğrudan doğruya--dan zebra balığı embriyo ve yaklaşımlar ve uzun ömürlü kültür artırmak ekili çeşitliliği genişletmek için birlikte yetişkin zebra balığı beyin primer hücre hazırlamak için hem bir etkili ve kullanımı kolay iletişim kuralı kurulan hücre tipleri. Buna ek olarak, biz embriyonik primer hücre ifade yapıları floresan organel işaretleri ile transfect için bir yordam mevcut. Böylece, hücresel türleri Morfoloji ve hücre altı yapıları korumak onların temel özellikleri farklı hücre tipleri yüksek zamansal ve mekansal çözünürlükte ile çözümlenebilir.

<table><tbody><tr><td>&lt;u&gt;Fish lines&lt;/u&gt;</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(&lt;em&gt;ptf1a&lt;/em&gt;:eGFP)jh1</td><td></td><td></td><td>stable transgenic line in which the enhancer of the zebrafish gene&lt;em&gt; ptf1a &lt;/em&gt;drives expression of the fluorescent protein EGFP (Parsons &lt;em&gt;et al&lt;/em&gt;., 2007)</td></tr><tr><td>Tg(XITubb:DsRed)zf148</td><td></td><td></td><td>stable transgenic line in which the&lt;em&gt; Xenopus &lt;/em&gt;neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&amp;nbsp; (Peri and N&amp;uuml;sslein-Volhard, 2008)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</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 &amp;deg;C temperature box and a 63X objective</td></tr><tr><td>epifluorescent microscope</td><td>Leica microsystems</td><td></td><td>model DM5500B, equipped with 28 &amp;deg;C temperature box and a 40X 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 &amp;deg;C)</td><td>Memmert</td><td></td><td>model incubator I</td></tr><tr><td>incubator for embryos (28 &amp;deg;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plasmids&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>pCS-DCX-tdTomato</td><td>K&amp;ouml;ster Lab</td><td># 1599</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-eGFP</td><td>K&amp;ouml;ster Lab</td><td># 7</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-H2B-mseCFP</td><td>K&amp;ouml;ster Lab</td><td># 2379</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-mClover</td><td>K&amp;ouml;ster Lab</td><td># 3865</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-MitoTag-YFP</td><td>K&amp;ouml;ster Lab</td><td># 2199</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-ss-RFP-KDEL</td><td>K&amp;ouml;ster Lab</td><td># 4330</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-VAMP1-mCitrine</td><td>K&amp;ouml;ster Lab</td><td># 2291</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.,&lt;/em&gt; 1994)</td></tr><tr><td>pSK-UAS:mCherry</td><td>K&amp;ouml;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&amp;ouml;ster lab. Plasmids are available upon request.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plastic and glass ware&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Falcon Cell Strainer (40 &amp;micro;m)</td><td>FALCON</td><td>REF 352340</td><td>distributed by BD Bioscience, used as &amp;ldquo;landing net&amp;rdquo; 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 &amp;micro;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&amp;auml;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 &amp;micro;l): 70762; Yellow (200 &amp;micro;l): 70760002; White (10 &amp;micro;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Chemicals and Reagents&lt;/u&gt;</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 1x PBS</td><td>Sigma-Aldrich</td><td>16005</td><td></td></tr><tr><td>4&#039;,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 &amp;alpha;-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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Enzymes&lt;/u&gt;</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 &amp;deg;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 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Medium and solutions for cell culture&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS (Dulbecco&#039;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>CO&lt;sub&gt;2&lt;/sub&gt;-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 100x</td><td>Gibco by Life Technologies</td><td>25030081</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>Leibovitz&#039;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 U/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

Zebra balığı embriyo şeffaf ve hızla böylece bir sağlam ve gelişmekte olan omurgalılar dinamik biyolojik süreçlerin mükemmel vivo içinde görüntüleme için izin Anne, dışarıda geliştirmek. Ancak, farklı hücre tipleri ve hücre altı yapıları türleri morfoloji ayrıntılı görüntüleme tüm bağlar sınırlıdır. Bu nedenle, etkili ve kullanımı kolay bir iletişim kuralı kültür canlı primer hücre için zebra balığı embriyo ve yetişkin doku kurduk.
Kısaca, dechorionated, deyolked, steril ve collagenase sahip tek kişilik hücrelerin ayrışmış 2 dpf zebra balığı embriyo vardır. Filtrasyon adımdan sonra primer hücre cam alt yemekleri kaplama ve birkaç gün için ekili. Taze kültürler, olduğu kadar uzun vadeli differenciated olanlar, yüksek çözünürlüklü confocal görüntüleme çalışmaları için kullanılabilir. Kültür çizgili miyositler ve poli-L-lizin kaplama üzerinde önemli varlık nöronlar ile farklı hücre türleri içerir. Özellikle etiket hücre altı yapıları için floresan marker proteinler tarafından plazmid DNA nöronlar da dahil olmak üzere farklı hücre tipleri, içine transfection sağlayan bir adım iletişim kuralı da kurduk. Böylece, operatör huzurunda tanımlanmış uyaranlara, karmaşık hücre davranış ve hücre içi birincil zebra balığı hücre dinamiği yüksek uzamsal ve zamansal çözünürlük ile değerlendirilebilir. Buna ek olarak, Yetişkin zebra balığı beyin kullanarak, açıklanan ayrılma tekniği gibi temel kodlamayla koşulları da yetişkin zebra balığı doku için iş göstermektedir.

Şekil 1G çizgili miyositler ve en bol olan nöron benzeri hücre kümeleri ile vahşi türü embriyo elde tipik bir kültürün iletilen bir ışık görüntü gösterir. Bazı hücre tipleri daha kolay tanımlamak için hücre türüne özgü ifade floresan bir protein ile transgenik bir çizgi olabilir (Şekil 1 H) kullanılır.
PCS2 + transfection-floresan protein (eGFP)15 wild türü embriyo sonuçlar 1 güçlü bir floresan sinyal primer hücre içine dap gelişmiş yeşil kodlama plazmid11 (Şekil 2A) dayalı. Tarafından transfecting pCS2 +-floresan organel işaretleri endoplazmik retikulum hedefli kırmızı floresan protein (ss-RFP-KDEL)16 veya mitokondri hedefli Sarı floresan protein (MitoTag-YFP)17, gibi kodlama plazmid dayalı 18, hücre altı yapıları (Şekil 2B, C) ayrıntılı olarak görüntülenir. En çok üç plazmid Co transfection sağlar aynı hücre19 MitoTag-YFP gibi Mikrotubul marker ile birlikte çeşitli floresan füzyon proteinlerin hücre altı yerelleştirme eşzamanlı analizi için insan Doublecortin (DCX) 20 kırmızı floresan protein tdTomato21 ' e erimiş ve nükleer marker histon H2B Camgöbeği bir floresan protein (H2B-mseCFP)22,23 (Şekil 2B) erimiş. Hücre altı yapıları da floresan protein24 mCitrine erimiş yüksek zamansal ve mekansal çözünürlüklü, örnek veziküller membran işareti. sivilce ilişkili membran proteini 1 olumlu hareketi (VAMP1) görüntüsü , 25 (Şekil 3). Zaman içinde morfolojik değişiklikleri kolayca kes26 (Şekil 2 g, ben) gibi parlak bir floresan protein ifade tarafından tüm cep telefonu etiketleme tarafından görülebilir. Ancak, her iki Gal4 sürücü taşıyan çift transgenik embriyo floresan protein mCherry27 UAS öğeye primer hücre erimiş kırmızı kodlama pBluescriptSK omurga dayalı bir plazmid başarılı transfection türetilmiş yapý ve bir UAS efektör inşa gösterir Elektroporasyon protokolü de Kombinatorik genetik (2E rakam) için uygundur. Ayrıca, sabit primer hücre kolayca ayirt ve organel özgü floresan boyalar (Şekil 2F, H) kullanarak boyanabilen.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57872/57872fig3.jpg"> Şekil 3: zamanlı hızlandırılmış görüntü hücre altı yapıları. Vahşi türü hücre (dap 1) adet-VAMP1-mCitrine ile transfected. Zaman sukut yazmak çerçevelerin seçili (1 kare her 1,68 alınmıştır s) gösterilir. Ok uçları ve parça renkler göre içinde üç ayrı VAMP1 pozitif veziküller hücre altı dinamikleri vurgulayın. Ölçek çubuğu 10 µm =. Zaman atlamalı mikroskobu tarama confocal lazer kullanılarak kaydedildi. <a href="https://www.jove.com/files/ftp_upload/57872/57872fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
Ayrıca yetişkin zebra balığı dokusu ile ayrılma teknik ve temel kültür koşulları uygulanabilir. Burada, vahşi türü zebra balığı yetişkin beyin dokusundan test ettik. Şekil 4A -D karmaşık nöronal benzeri ağa başlangıçta enkaz kaplı kültürünün ilerici geliştirme gösterir. Yetişkin beyin transgene Tg taşıyan bir Transgenik balık kullanarak (XITubb: DsRed) zf14828tamamen farklı, DsRed ifade nöronlar (Şekil 4E) yakın ayrıntılı olarak incelendiğinde.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57872/57872fig4.jpg"> Şekil 4: yetişkin zebra balığı beynin birincil hücre kültürü. Primer Hücre parlak alan görüntüleri elde edilen yetişkin zebra balığı beyin (a)1 dap, (B) 3 dap, (C) 5 dap ve (D) 8 dap. 1-3 dap, ölü hücreleri ve doku enkaz iken tek veya kümelenmiş nöronal hücre daha belirgin hale kaybolur. 3 dap, nöronal hücre ilk kısa neurites kurmaya başlar. 5'ten itibaren dap, bir ağ ile yüz-in de uzun neurites oluşumu gözlemlemek mümkündür. Ölçek çubukları = 50 µm. hücreleri poli-L-lizin-kaplı bir 96-şey plaka kültürlü. 7 (E) kültürlü hücreleri transgene Tg ifade bir Transgenik balık yetişkin beyinden dap (XITubb: DsRed) zf148. DsRed farklı nöronlar28ifade edilir. Ölçek çubuğu = 100 µm. hücreleri bir poli-L-lizin-kaplı 24-şey tabak içinde kültürlü. Tüm görüntüleri bir epifluorescent mikroskobu tarafından elde. <a href="https://www.jove.com/files/ftp_upload/57872/57872fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

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

Culture and Transfection of Zebrafish Primary Cells

Biz birincil hücre kültürleri zebra balığı embriyo transfection ve canlı hücre görüntüleme gibi primer hücre--dan yetişkin zebra balığı beyin hazırlamak için bir protokol için hazırlanması için etkili ve kullanımı kolay bir protokol mevcut.

Kültür ve zebra balığı primer hücre Transfection

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

culture-and-transfection-of-zebrafish-primary-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K Görüntüleme.  Braunschweig University of Technology. Biz birincil hücre kültürleri zebra balığı embriyo transfection ve canlı hücre görüntüleme gibi primer hücre--dan yetişkin zebra balığı beyin hazırlamak için bir protokol için hazırlanması için etkili ve kullanımı kolay bir protokol mevcut.

Video: Culture and Transfection of Zebrafish Primary Cells

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Zebra balığı Keratosit Eksplantlarındaki Deri Yara İyileşmesi Toplu Hücre Göç ve reepithelialization Eğitim için

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Gelişim Biyolojisi

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

Embriyonik Zebra balığı İnsan Kanser Hücrelerinin İnvazif Davranış

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Kanser Araştırmaları

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Yetişkin balığı beyin türevli Neurospheres İzolasyon ve Kültürü

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

İzolasyon ve Zebra balığı Embriyolar gelen Tek Hücre Karakterizasyonu

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Multiphoton Mikroskopi Canlı hücre Görüntüleme için Yetişkin Transgenik Zebra balığı Retina Eksplantlarından Kültür

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. 
Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Here, we present a protocol to establish versatile orthotopic and ectopic zebrafish xenograft models for ocular melanoma to assess the growth kinetics of the primary tumor, dissemination, extravasation and distant, peri-vascular metastasis formation and the effect of chemical inhibition thereon.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Zebra balığı erken embriyo ve tümör hücresel elektrik aktivitesinin görselleştirme

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Embriyonik ve Karaciğer Zebra Balığı Hücre Hatlarının Sferoidlerinin 3D Kültür Yöntemi

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Çevre

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Zebra Balığı Tol2 Sistemi: Modüler ve Esnek Ağ Geçidi Tabanlı Transgenez Yaklaşımı

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

<meta charset="utf8"></head><body>Rejenerasyon ve Gelişim Araştırmaları için Sağlam Zebra Balığı Hücre Nakli

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>Yazar Spotlight: Gelişim ve Rejenerasyon Sırasında Zebra Balığı Sinir Sisteminde Hücresel Bağlantının Haritalanması

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Embriyonik ve Larva Zebra Balıklarında Yüksek Çözünürlüklü Hücre Nakli

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...