Mükemmel balık yetiştiriciliği için Bayan Paula Roy ve Steve Ekker pDB600 plazmid tür hediye için teşekkür ederim. Bu çalışmalar NHGRI SF bir hibe ile finanse edilmiştir.

1. Seçme ve test etmek için korunmuş dizilerinin klonlanması <ol><li> Bir ilk fonksiyonel testleri için potansiyel düzenleyici dizileri tanımlamak gerekir. Biz dizileri seçmek için bir kriter olarak evrimsel bir sıralama koruma güveniyor ve en sık UCSC Genom tarayıcı (http://genome.ucsc.edu/cgi-bin/hgGateway) bir parça olarak erişilebilir algoritma PhastCons kullanabilirsiniz. Ancak, türler arasında sırası koruma değerlendirmek için pek çok diğer algoritmaları vardır. </li><li> Bir kere biz bizim dizileri seçtiniz, biz her genomik DNA sırasını yükseltmek için primerler dizayn. Astarlar korunmuş bölgenin her iki tarafında bulunan artı 30 veya daha fazla baz çifti yükseltmek için seçilmiş olmalıdır. </li><li> Seçilen dizilerinin amplifikasyonu için Takara LA TaqTM sistemi kullanır. Diğer polimerazları çalışır, ama amplifikasyon sırasında tanıtılan hataları şansını en aza indirmek için, redaksiyon yeteneğine sahip bir enzim içeren bir karışım kullanmak önemlidir. Biz agaroz jel elektroforezi ile Amplikon boyutu olun. </li><li> Biz PCR 8/GW/TOPO TA Klonlama Kiti (Invitrogen) attL rekombinasyon siteleri içeren bir giriş vektörü PCR ürünü clone Gateway giriş klon oluşturmak için kullanın. Klonlama tepki taze PCR ürünü ile daha verimli, böylece bir gün içinde PCR klonlama gerçekleştirmek için en iyisidir. DH5α suşları Dönüşüm spectinomycin direnç seçimi yapılır. </li><li> Biz insert serbest EcoRI ile plazmid ~ 500ng sindirim TA klonlama ürün doğrulamak ve agaroz jel elektroforezi ile ekleme boyutunu onaylayın. Biz ekleme sırası ve yönünü kontrol etmek için sıralama önerilir; amplifikasyonu için kullanılan primerler sıralaması için de kullanılıyor olabilir. </li><li> Giriş klon olmayan kodlama korunmuş dizisi Gateway LR Clonase II enzim Mix (Invitrogen) kullanarak Ağ Geçidi hazır hedef vektörü, pGW_cfosGFP1, LR rekombinasyon mekik. Dönüşüm DH5α suşlarının ampisilin direnç seçimi yapılır. </li><li> Biz insert serbest EcoRV ile plazmid ~ 500ng sindirim LR rekombinasyon ürünü doğrulamak ve agaroz jel elektroforezi ile ekleme boyutunu onaylayın. Doğru bir klon tespit edildikten sonra, biz Qiagen HiSpeed ​​kullanarak plazmid DNA ® Plazmid Midi Kit hazırlar. Üreticinin protokollere göre daha QIAquick PCR Arıtma Kit kullanarak plazmid arındırmak ve 30 mcL RNaz ücretsiz su ile DNA Zehir. Plazmid 125 mcL ng / 4 saklanan ° C önceden enjeksiyonları bir konsantrasyona seyreltilir. </li><li> RNA kodlama fonksiyonel Tol2 transposase enzim pDB600 plasmid2 in vitro transkripsiyonu. Biz Qiagen Midi-Prep kiti kullanılarak plazmid arındırmak, daha sonra 10-20 mg XbaI linearize. MRNA sentezini mMESSAGE mMACHINE Kiti protokolü (Ambion) yürütülmektedir. </li><li> Biz arındırmak ve kiti talimatlarına göre RNA hızlandırabilir. Biz RNaz ücretsiz su ~ 1μg/μl nihai bir konsantrasyon, ya da tek bir reaksiyon için 20 ul RNA tekrar süspansiyon ve UV spektrofotometresi ile ölçmek. Ayrıca, tam uzunlukta transkripsiyon doğrulamak için agaroz jel elektroforezi ile RNA ~ 1μg analiz. Bu analiz için standart TAE veya TBE jel yeterli olmasına rağmen, transkripsiyon kiti ile denatüre örnek tampon kiti talimatlarına göre kullanılmalıdır. </li><li> Biz, bilinen bir plazmid (Şekil 1) enjeksiyonları yaparak etkinlik için, transposase RNA her yeni toplu test edin. Denetlemeden sonra, RNA küçük alikotları içine bölünmüş ve -80 saklanan ° C, tekrarlanan çözülme ve bireysel alikotları, tekrar soğutulmasından kaçınmak için. <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="Şekil 1" /> Şekil 1. Zebra balığı embriyo fare Sox10 arttırıcı ile kontrol (Üst) aydınlık (Alt) GFP floresan görüntü olarak enjekte edildi. Transposase RNA Her yeni toplu etkinlik için test edilir. </li></ol> 2. Mozaik transgenik analizi için zebrafish embriyoların Mikroenjeksiyon <ol><li> Biz sağlam chorions nüfuz oldukça keskin konik güçlü bir ipucu verim için tasarlanmış bir program ile, 1.2 mm OD filament kılcal cam bir Sutter P-97 Mikropipet Çektirme mikroenjeksiyon için iğne çekin. Biz temiz bir jilet ve bir mikrometre slayt kullanarak, dış çapı yaklaşık 15 mikron stereomikroskop altında elle ipuçları bölünürler. Iğneler gün enjeksiyonlar önce yapılmış ve temiz tutmak için kapalı bir elinde tabak içinde saklanan olabilir. </li><li> Biz, 14 saat ışık altında standart conditions3, düzenli bir ışık karanlık döngüsünde zebrafish koruyacağız. Gün önce performans microinjections, biz küçük bir yetiştirme tankları, bir temel tank oluşan her bir oluklu eklemek ve plastik bir kapak zamanlanmış çiftleşmelerin balık kurdu. Biz üç kadın veya iki erkek t her yerdeparalel sıralar halinde Ank. </li><li> Microinjections sabahı, ışık döngüsü başlar kısa bir süre sonra, erkek ve kadınlarda yumurta üretimi için temiz bir sistem arıtılmış su birleştirerek başlar. Balık sık sık kontrol edin ve birkaç dakika içinde döşeme yumurta toplamak iyi senkronize toplu almak için önemlidir. Yumurta üretimi, sabah boyunca taze balık grupları birleştirmek için devam ederek, 2-3 saat boyunca muhafaza edilebilir. </li><li> Geniş bir delikli, 5 1 / 4 &quot;cam Pasteur pipeti bir lateks ampul ile donatılmış, kısmen Embriyo Medium2 dolu 50 embriyoların gruplar halinde 60 x 15 mm Petri kapları, toplanan embriyolar sıralamak ve işaretlemek zaman toplanan Her çanak kapağı. </li><li> Balık döşeme bir kez başladıktan sonra, biz, buz üzerinde bir mikrofuge&#39;de tüp içinde aşağıdaki karıştırılarak taze enjeksiyon çözelti hazırlamak: <table border="1"><tbody><tr><td> Bileşen </td><td></td></tr><tr><td> Transposon plazmid stok (125ng/μl) </td><td> 1μl </td></tr><tr><td> Transposase RNA stok (175ng/μl) </td><td> 1μl </td></tr><tr><td> Fenol kırmızı stok (H 2 O 2%) </td><td> 0.5μl </td></tr><tr><td> RNaz ücretsiz su </td><td> 2.5μl </td></tr></tbody></table> Enjeksiyon iğneleri, yemekler tutarak koydu ve geniş ucuna her iğne enjeksiyon solüsyonu 500 nl damla pipetleme tarafından doldurulacaktır. Ucu kapiller eylem yoluyla sıvı çekilir sonra, ek çözüm 1.5-2 ul toplam eklenebilir. Biz her enjeksiyon çözümü için en az iki iğne hazırlar. </li><li> Dolu iğne yapılandırılmış ve üreticinin talimatlarına göre N2 tankına bağlı bir Pnömatik Pico-Pompa veya benzeri basıncı enjektör, iğne tutucu düzenlenen el yüklenir. Biz bir mikrometre slayt, mineral yağ atılır damlacıkları çapını ölçerek enjeksiyon hacimleri kalibre edin. Tipik olarak, 20 psi basınç ile 120 ms bir enjeksiyon süresi yaklaşık 1 nl bir damlacık verecektir, ancak iğne çapı küçük farklılıklar bu parametreleri etkileyecek ve kalibrasyonu iğneler arasında gerekli olabilir. </li><li> Enjeksiyonları, 6-10X büyütme stereomicroscope altında yapılmaktadır. Biz ince forseps embriyo yerde tutun koryon ve bir hücre veya iki hücre erken aşamada bir embriyo sarısı içine sabit bir basınç ile enjeksiyon iğnesi itmek için bir çift kullanın. İdeal olarak, biz sadece blastomer altında sarısı iğne ucu yerleştirin. Enjeksiyon solüsyonu yaklaşık 1 nl kovdu ve iğne geri çekilir. Her biz ≥ 200 embriyolar enjekte oluşturmak için. Uninjected kontrol embriyoların bir çanak kaydetmek için, toplanan embriyoların her kavrama de çok önemlidir. </li><li> Enjeksiyonları tamamlanmıştır sonra, döllenmemiş yumurta, hasarlı embriyolar ve başarısız enjeksiyonları (blastomerlere hiçbir fenol kırmızısı embriyo) kaldırarak embriyoların sıralayabilirsiniz. Daha sonra için 28.5 ° C uygun zaman kadar gözlemler embriyolar embriyo ortamda kuluçkaya yatmaktadır. </li></ol> 3. Mozaik ekspresyonu analizi <ol><li> Uygun bir kuluçka süresini takiben, floresan enjekte edilen embriyolar inceleyin. Zamanlama endojen zebrafish genin normal ifade desenini bağlıdır, ancak genellikle ilk 5 gün gübreleme yoluyla günlük bak. </li><li> Biz Epifloresans için takılan bir Olympus MVX10 Makroskop&#39;ta floresan embriyoların incelemek, ancak herhangi bir benzer bir mikroskop çalışma düşük güç görüntüleme yeteneğine sahip olacak. Ölen ya da ilk gün anormal şekilde gelişmiş çok sayıda embriyo varsa, normal bakarsanız bakın bu kavrama uninjected kontrolleri bakmak. Kontrolleri normal bakarsanız, en sık karşılaşılan sorun, enjekte edilen DNA yetersiz saflık. Ayrıca, embriyoların erken dönemde toplam tutarı enjekte plazmid duyarlı, bu yüzden miktarının yanlış olduğunu ve çok fazla DNA enjekte olduğunu mümkündür. </li><li> Embriyolar incelerken, hepsi enjekte inşa mozaik olacağını aklınızdan çıkarmayın. Bu ifade beklenen etki alanı küçük, özellikle de tüm embriyoların dikkatli bakmak gerekli olacaktır. Ayrıca ifade dinamik olabileceğini unutmayın; güçlü floresan gün 1 gün 2, ve ifade etmek için ilk 3 gün günde 4 şey gösterebilir negatif embriyolar kaybolabilir. </li><li> Biz daha geniş bir ifade ile bazı embriyolar seçin ve photomicroscopy floresan desen belge bu. Gözlemlerin 5 gün sonra, embriyoların tesisine iade edilmelidir ve bir hisse senedi olarak gündeme. Birkaç ay içerisinde, yetişkinler transgenlerin germline iletimi için taranmalıdır. </li></ol> 4. Sonuçlar Biz geniş bir v bakınbağlı olarak analiz edilen artırıcı unsuru desen ve ifade düzeyleri ariety. Biz genellikle çok doku spesifik ekspresyonu ilgilendi ve genellikle iskelet ifade genler üzerinde duruluyor. ve genellikle iskelet genlerin üzerinde duruluyor. Şekil 2, kıkırdak ve kemik ifade düzenleyen arttırıcılar ile enjekte edilen embriyolar gözlenen mozaik ekspresyonu örnekleri gösterir. Son olarak, test dizilerinin önemli bir kısmının doku spesifik ifade düzenleyen yok. Bunlar için, çoğu kez çok az floresan, embriyo başına belki birkaç dağınık hücreleri bakın. Daha az sıklıkta, ama sadece iki ortak siteleri ektopik ifade, epidermis ve iskelet kası (Şekil 3), floresan görebilirsiniz. <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="Şekil 2" /> Şekil 2. Enjekte edilen embriyolar gözlenen kıkırdak ve kemik ekspresyonu örneği. <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="Şekil 3" /> Şekil 3. Gen ve ifade düzenlenmesi arttırıcı göreli konumu arasında çok az bir korelasyon vardır. Test dizilerinin önemli bir bölümü, doku spesifik ifade düzenleyen yok . Bunlar genellikle çok az floresan veya ektopik ifade için iki ortak olabilir: epidermis ve iskelet kası. (Üst) aydınlık görüntü (Alt) GFP floresan görüntü.

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</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. , (1995).</li></ol>

Biz potansiyel arttırıcılar zebrafish transgenesis kullanarak etkin bir şekilde analiz için laboratuarda kullanılan yaklaşım göstermiştir. Çoğu zaman, biz insan genleri ile ilişkili doku-spesifik arttırıcılar bakmak için bu yaklaşımı kullanmak. Bariz dizi homoloji olmamasına rağmen çoğu zaman insan ve balık olmayan kodlama dizileri, bu yaklaşım bize ilgi genler için arttırıcılar belirlenmesinde genellikle başarılı olduğunu bulmak. Arasındaki Başarı için kritik faktörler, plazmid D...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
<td>&nbsp;</td>
<tr>
<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
<tr>
<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
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<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
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<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
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<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
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<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

, Pek çok diğer türler ile birlikte insan genom dizisinin tamamlanması, belirli bir işlevi olmayan kodlama dizileri vermenin zorluk ön plana çıkarmıştır. Bir genomun olmayan kodlama fraksiyonu tarafından yürütülen önemli bir fonksiyonu gen transkripsiyonu düzenleyen, ancak genel olarak birincil DNA dizisi cis-düzenleyici unsurları tahmin etmek için etkili yöntemler vardır. Biz zebrafish transgenesis aracılığıyla potansiyel cis-düzenleyici unsurlar işlevsel değerlendirmek için verimli bir protokol geliştirmiştir. Bizim yaklaşımımız, mekansal ve zamansal gen regülasyonu hakkında bilgi sağlar, gelişimsel olarak önemli genler hücre kültür teknikleri üzerinde önemli avantajlar sunuyor. Tersine, transgenik farelerin benzer deneyler daha hızlı ve daha az pahalı ve rutin insan genomu izole dizileri uygulayabilirsiniz. Burada sıra koruma ve klonlama dizileri için bizim protokol tabanlı ve zebrafish embriyolar içine microinjecting test etmek için eleman seçerken bizim yaklaşım göstermektedir.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Biz, gelişimsel olarak düzenlenir genleri potansiyel artırıcı unsurları bulma ve mozaik zebrafish transgenesis fonksiyonu değerlendirmede bizim yaklaşımımız göstermektedir.

Mozaik Zebra balığı Transgenesis Enhancer Diziler değerlendirilmesi için

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K Görüntüleme.  University of Pennsylvania. Biz, gelişimsel olarak düzenlenir genleri potansiyel artırıcı unsurları bulma ve mozaik zebrafish transgenesis fonksiyonu değerlendirmede bizim yaklaşımımız göstermektedir.

Video: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebra balığı Beyin Ventrikül Enjeksiyon

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Biyoloji

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Necromenic nematodu Mikroenjeksiyon Gen demonte ve Transgenesis aracılığı ile Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Yenileyici Yetişkin Zebra balığı Tail Fin içine Morpholinos in vivo olarak Elektroporasyon

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Mozaik Yetişkin Canlı Görüntüleme Domates / GFP-FLP / FRT Yöntemi<em&gt; Drosophila</em&gt; Fotoreseptör Hücreler

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUT Hız Sedimentasyon kullanma spermatogenic Hücre Tiplerinin Ayrılması

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Dinamik Ca Otomatik Analizi<sup&gt; 2 +</supGörüntü Dizilerinde&gt; Sinyaller

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Değerlendirilmesi Hücre Mechanosensing ve Durotaxis için Basitleştirilmiş Sistemi<em&gt; İn Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...