Bu eser (LH DK093625 ve DK069909 ve DK047757 JHL için) NIH tarafından desteklenen ve VA (Başarı Ödülü JHL için I01BX000820). Biz gerekli desteği sağlamak için Pennsylvania Zebra balığı Core Üniversitesi&#39;nden Dr. Michael Paketi ve Dr Jie He teşekkür etmek istiyorum.

NOT: Etik Açıklama: Tüm zebrabalıkları deneyler Eastern Virginia Medical School Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Morfolino Hazırlık <ol><li> (Şekil 1A), üreticinin talimatlarına uygun olarak söz konusu gen için tasarım ve sentez için engelleme (AUG-) ve ek yeri engelleyici (Splice-), anti-sens morfolino (MO) oligonükleotidleri. Tablo 1&#39;de üreticinin bilgilerine bakın lütfen. NOT: MOs cam şişelerde liyofilize hisse senetleri olarak sevk edilir. </li><li> 25 ug / ml&#39;lik bir son konsantrasyona kadar MOS yeniden askıya cam şişelere yüksek dereceli steril su ekle. Oligonükleotid tamamen eriyene emin olun. Bazı katı kalıntılar ise, 5 kısaca dk ve vorteks 10 için 65 ° C&#39;de stok oligonükleotid çözeltisi içeren flakon ısı. </li><li> Oda sıcaklığında MO stok solüsyonu saklayın. 4 ° C veya -20 ° C&#39;de saklayın etmeyinC daha düşük sıcaklıklar MO oligonükleotid konteyner duvarına bağlamak için neden olabilir çünkü. Bir spektrofotometre kullanılarak stok çözeltisinin konsantrasyonunu ölçmek, yeni bir MO stok çözeltisi yapılan her zaman (Tablo1 bakınız). </li><li> Istenen doz, yüksek dereceli steril su ile stok çözelti seyreltilerek enjeksiyonu gününde, MO çalışma çözeltisi hazırlayın. % 0.05&#39;lik bir konsantrasyon elde etmek üzere% 0.5 fenol-kırmızı ekleyin. Örneğin, 15 ng / nL bir konsantrasyon ile 5 ul çalışma solüsyon elde etmek amacıyla 3 ul MO stok çözeltisi ve 0.5 ul% 0.5 fenol kırmızısı su, 1.5 ul ekle. NOT: Bu çalışma konsantrasyonu ile enjeksiyon her damla (500 pl) MO 7.5 ng içerir ve iki damla MO 15 ng içerir. </li></ol> Enjeksiyon Aparatı 2. Hazırlık <ol><li> Satınalma cam (Ayrıntılı bilgi için bakınız Tablo 1 lütfen) iğneler önceden çekti. Alternatif olarak, cam enjeksiyon iğnesi wi çekinBir iğne çektirmesi inci. </li><li> Hava kompresörü açın ve 50 psi basınç ayarı ayarlayın. Diseksiyon mikroskobu ışık kaynağı ve Pico mikro-enjeksiyon pompası açın ve aşağıdaki gibi ayarları: basıncı 20 psi tutun, basınç 10 psi, yolluk 2,5 ve 100 mikro saniye aralığı süresi değerini çıkarma. </li><li> Enjeksiyon çözeltisi 5 ul camını önceden çekti iğne yükleyin ve ucu aşağı bakacak şekilde dikey konumda iğne yerleştirin. Tüm çözüm görünür hava kabarcıkları ile iğne ucu ulaşana kadar bekleyin. Mikroskop yanında uygun bir konumda iğne tutucu yerleştirin ve tutucu içine cam iğne yerleştirin. 45 derece enjeksiyon açısını ayarlayın. </li><li> Yüksek sahneden, mikroskop altında görünümü içine iğne ucu getir, ve ucu ince bölgeye odaklanmak. İnce nokta cımbız ile iğne ucu kırın. </li><li> Bir cetvel ve mikroskop altında yan 0,15 mm tarafının iç çapa sahip bir kılcal tüp yerleştirin;bir sıvı sütununu yapmak için kılcal borunun bir ucuna solüsyonu enjekte edilir. 1 mm uzunluk sıvı kolon başına her 17 damla ulaşmak için çıkarma zamanı ayarlayın, daha sonra her damla hacmi 1 nl sıvı sütunun = π x yarıçapı 2 x uzunluk (damla hacmi () damla / sayımı () &#39;dir. Not: 0.1 mm bir çapı olan bir damla 500 ul bir enjeksiyon hacmi (damla hacmi = 4/3 x π X yarıçapı 3) elde edilir. </li></ol> Morpholinos 3. döllenmiş Zebra balığı Embriyolar hazırlanması ve Enjeksiyon <ol><li> Standart Zebra balığı yetiştiriciliği ve bakım için yayınlanan protokollere göre transgenik zebra balığı üreyen: Tg (GFP wt1b) ayarlayın. Doğal yumurtlama sonra embriyolar toplamak ve E3 su ile dolu bir 10 cm Petri için (5 mM NaCI, 0.17mm KCI, 0.33 mM CaCl2, 0.33 mM MgSO 4) &#39;de muhafaza edin. Hemen 13 MO enjeksiyon başlatın. Mikroskop altında embriyo morfolojisi izlemek ve emin olun MO enjeksiyon tek olanHücre sahne, ya da dört-hücre aşamasında daha sonra. <ol><li> Ile 10 cm&#39;lik bir petri tabağına embriyoların transferi olukları agar kama şeklinde tasarlanmış olmasıdır. Ekstra E3 suyu aspire ve yavaşça olukları içine embriyolar basın. </li><li> Mikroskop altında 1-hücre aşaması embriyo görselleştirmek için mikropipet ile embriyolar işleyin. Yumurta sarısı (1 damla = 500 ul) içine MO bir iki damla enjekte koryon ve yumurta sarısı nüfuz. E3 su ile 10 cm&#39;lik petri enjekte embriyolar transfer ve 28.5 ° C&#39;de inkübe. </li></ol></li><li> Enjeksiyondan sonra, ölü embriyolar kaldırmak ve enjekte edilen embriyoların sayısını kaydetmek. Çanak, her 24 ila 48 saat içinde E3 suyu değiştirin. </li></ol> 4. Fenotip Kurtarma Deneyler <ol><li> Kurtarma MO dayanıklı nedenle, farklı bir birincil baz çifti yapısı (genellikle 3-7 baz çifti uyumsuzlukları) sahiptir ve başka türlerden, bir ortolog seçin. Örneğin, bu deneyde, seçtikfare fare Wnt5a mRNA dizisi Zebra balığı diziden farklı olduğu için. </li><li> Ileri primer translasyon sitesi ve mRNA kodlama bölgesinin sonuna yakın bir ters primer olarak başlayan bir primer tasarımı. Ileri primer dizisinin 5&#39;-ucunda, bir T7 yükseltici seri ekleyin. Bu deneyde, aşağıdaki primer dizileri kullanılır; ileri: 5&#39;taa tac gac tca cta etiketi gga cta TGA tgc tgc TGA agc TGA a- 3 &#39;ve ters: 5&#39;tca CTT DHA gac gta ctg gtc- 3&#39;. </li><li> Yukarıda tasarlanmış bir primer grubu (her bir primerden 0.5 uM) ve Aşağıdaki PCR programı ile fare Wnt5a cDNA sekansını ihtiva eden 0.1 ug şablon DNA ile birlikte 50 ul Polimeraz Zincir Reaksiyonu (PCR) yapın: 5 dakika boyunca 95 ° C &#39;de 1 döngü; 95 ° C (30 saniye), 58.5 ° C (30 saniye), 72 ° C (1 dakika) 35 döngü; ve son uzatma adımı, 7 dakika boyunca 72 ° C&#39;de dayanıklılığı. </li><li> T bitirdikten sonraO döngüsü, bant doğru boyutta olduğundan emin olmak için% 1 agaroz jel üzerinde PCR ürünü 2 ul çalıştırın. Üreticinin talimatlarına uygun olarak bir PCR saflaştırma kiti ile PCR ürünü saflaştırılır. Bir spektrofotometre ile DNA konsantrasyonu kontrol edin. -20 ° C&#39;de saflaştırılmış PCR ürünü depolamak ya da doğrudan bir sonraki aşamada başlıklı RNA transkripsiyonu kullanın. </li><li> Üreticinin talimatlarına göre bir başlıklı bir RNA sentez takımı ile mRNA başlıklı in vitro sentez. 20 ul RNazsız su içinde seyreltilir ve mRNA konsantrasyonunu belirlemek. -80 ° C de, RNA ve mağaza alikosu. </li><li> Enjeksiyon gününde, RNaz içermeyen steril su ile bir mRNA çalışma çözeltisi hazırlayın. 40 ug RNA, spesifik ya da toksik etkilere yol açmaz olan en yüksek doz genel olarak bir RNA olduğuna dikkat edin. Buz üzerinde çalışma çözüm tutun. MO ile embriyo ve daha sonra kurtarma mRNA, enjekte veya birlikte hem eş-enjekte. Örneğin, mRNA HAZIRLIK konsantrasyonuHer bir enjeksiyonun aşama 4.5 ed 0.08 ug / ul son konsantrasyon için RNazsız su içinde 4.33 ul mRNA (0.4 ug), 0.67 ul, 0.6 ug / ml, sonra bir damla (500 ul) 40 ug içeren mRNA. Aşama 3&#39;te tarif edildiği gibi MO hazırlayın. </li></ol> Floresan Görüntüleme Enjekte Embriyolar 5. hazırlanması <ol><li> 28.5 ° CO kuluçkalandıktan sonra / N, floresan mikroskopi kullanılarak pronephros yapının gözlenmesini etki melanization önlemek için E3 su ile% 0.003, N-feniltiyoüre (PTU) ekleyin. Erken embriyo gelişimini etkiler çünkü Ancak, gastrulasyon önce PTU eklemeyin. </li><li> 48 hpf, elle ince cımbız ile embriyolar dechorionate. Bir ışık diseksiyon mikroskobu altında 48 ve 72 hpf embriyoların görüntüleri çekmek. NOT: Bu resimler anestezi olmadan alınabilir. </li><li> , Flöresanlı mikroskop altında görüntüleme yerleştirerek embriyolar anestezi için yaklaşık 10 dakika önce160 ug / ml tampon Tricaine içeren 10 cm&#39;lik bir petri kabı. 5-10 dakika bekleyin, sonra hafifçe hareket etmeyecek ve bu nedenle anestezi çalıştığını onaylamak için zebrafish dokunun. </li><li> Embriyolar anestezi sonra, (Tablo 1 bakınız)% 3 metil selüloz onları monte ve onları mikroskop altında yüzüstü pozisyonda yönlendirmek. </li><li> Bir floresan mikroskop altında pronephros yapısını dikkate alınmalıdır (Tablo 1 bakınız) ve farklı büyütme (10X ve 20X) ile fotoğraf çekmek. </li></ol>

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The authors have nothing to disclose.

Polikistik böbrek hastalığı (PKD) insanlarda son dönem böbrek hastalığının önde gelen nedenlerinden biridir ve ilerici kist oluşumu, böbrek genişlemesi, ve anormal tübül geliştirme 14 ile karakterizedir. Otozomal dominant PKD (polikistik böbrek), genetik hastalık olan ya PKD1 mutasyonu, polikistin-1 (PC1) veya PKD2 kodlayan polikistin-2 (PC2) kodlayan, polikistik böbrek sonuçlanır. Diğer birçok gen, birincil cilium bulunan proteinleri kodlayan özellikle, böbrek kist gelişimi dahil o...

Balığı (Danio rerio) embriyo yaygın böbrek gelişim ve polikistik böbrek hastalığı çalışmak için bir model olarak kullanılmıştır. Orada bir hayvan modeli olarak zebrafish kullanarak pek çok avantajı vardır: genetik etkileşimlerin incelenmesinde fizibilite protein demonte için antisens morpholinos (MO) kullanma yeteneği fırsat hızlı bir şekilde embriyo sayıda tahlil edilmesi ve organ fenotipleri görüntüleme kolaylığı larva 1 yaşayan. pronephros omurgalılarda geliştirmek için ilk böbrek ve larva zebrafish 2 işlevseldir. zebra balığı pronephros yapısının memeli metanephros nispeten basit karşılaştırıldığında, üçüncü ve son böbrek, memelilerde geliştirmektir. nefron zor tek nefron yapısını gözlemlemek için yapım, 500,000-1,000,000 nefron 3,4 ve yaklaşık 13.000 nefron 5 olan her bir fare böbrek arasındaki içeren her insan böbreği ile, böbrek çalışma birimi in insan ya da fare böbrekler. zebra balığı iki nefron vardır ve her zebra balığı nefron glomerülleri ve fareler ve insanlarda 6 ve benzeri özelleştirilmiş böbrek hücre tiplerinin tübüllerde bulunan tüm ana bileşenleri içerir. Kapalı bir sistem 7 çünkü böyle Xenopus gibi diğer omurgalı modellere göre, zebra balığı nefron daha yakından memeli nefron benzer. Son yıllarda, zebra balığı genom genetik araçlar, geniş mutan kaynakları ve zebra balığı modelleri transgenik raportör hatları koleksiyonları geniş giriş sağlayan dizilenmiştir. 12-72 saat sonra döllenme (hpf) arasında Zebra balığı pronephros formları şeffaf embriyolar kolayca görülebilir. Wilm tümörü proteini WT1 böbrek gelişimi için önemli bir faktördür. Wt1b promoteri Tg kontrolü altında yeşil floresan protein (GFP) ifade eden transjenik zebrabalıkları hatları (wt1b GFP) GFP Expre göstermektedirsalgılanması, özellikle 17 hpf 8 başlayarak, Zebra balığı embriyolarının Pronephric bölgelerde yer. Nefronofitizi (NPHP), otozomal resesif kistik böbrek hastalığı, NPHP genlerinin 9 mutasyonlar neden olur. (Wt1b GFP) morfolino tarafından demonte NPHP4 Tg kist oluşumuna yol açtı. Balık 10 Bu nedenle, bu transjenik balık böbrek gelişimi esnasında böbrek yapıları ve kist oluşumunun gözlenmesi için uygun bir modeldir. Daha da önemlisi, böbrek gelişim modülatörlerinin etkisi, zaman ve iş gücü verimli bir şekilde, bu suşu kullanılarak incelenebilir. Gen modülasyonu sonrası böbrek kist oluşumuna görselleştirmek için bir model olarak, balık: Bizim kağıt Tg (GFP wt1b) kullanımını tarif etmektedir. Biz zebrafish Wnt5a geni yıkmak için başlangıç ​​ve bağlayıcı sitesi, anti-duyu MOS kullanılır. Wnt5a çeşitli organ ve hücre sonrası gelişiminde önemli bir rol oynar, Wnt ailesinin bir kanonik salgılanan bir glikoprotein olup,Fonksiyon 11. Wnt5a renal tübüler uzama esnasında yönlendirilmiş hücre bölünmesi bir rol oynadığı tespit edilmiştir düzlemsel hücre polarite (PCP) yol da dahil olmak üzere olmayan doğal Wnt yollar üzerinden çalışır. Wnt5a, bundan başka Vangl2 stabilite 12 teşvik VANGL düzlemsel hücre kutup protein 2 (Vangl2) ile bir kompleks oluşturarak Wnt5a sinyal çeviren tirozin kinaz benzeri reseptör (Ryk) ile bir kompleks oluşturarak Wnt / PCP yolu düzenler. PCP yolunda kusurlar rastgele hücre bölünmesi sonucu ve böbrek kist oluşumuna neden olabilir. Wnt5a demonte aşağıdaki böbrek kist oluşumunu gözlemlemek için Zebra balığı çizgi: Biz Tg (GFP wt1b) kullanılır. Tg (wt1b: GFP) Zebra balığı modeli canlı görüntüleme ve böbrek yapısının zamanında gözlem izin verir. Wnt5a demonte sonra, böbrek kist oluşumu 24 hpf başlayan bulundu; 72 hpf, kistler glomeruli ve proksimal tübüllerde bulunamadı. Bu yöntem aynı zamanda s kullanılabilirböbrek kist oluşumuna neden olabilir creen diğer genler.

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		<tr height="21">
			<td height="21" style="height:21px;width:221px;">0.5% Phenol-Red</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P0290</td>
			<td style="width:491px;">Color indicator for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Morpholino</td>
			<td style="width:229px;">Genn Tools, LLC</td>
			<td style="width:195px;">(customized)</td>
			<td>Customized designed to the gene of interest</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Pre-pulled needle</td>
			<td style="width:229px;">Tritech Research</td>
			<td style="width:195px;">MINJ-PP</td>
			<td style="width:491px;">If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">T7 mMessage Kit</td>
			<td style="width:229px;">Ambion</td>
			<td style="width:195px;">1344</td>
			<td>For in vitro transcription to make capped mRNA for rescue experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">N-Phenylthiourea</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P7629</td>
			<td>For prevention of melanization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tricaine</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">A5040</td>
			<td>Also called ethyl 3-aminobenzoate, for zebrafish anesthesia</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Methyl Cellulose</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">M-0387</td>
			<td>For position of zebrafish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">QIAquick PCR Purification Kit&#160;</td>
			<td style="width:229px;">Qiagen</td>
			<td style="width:195px;">28104</td>
			<td>For purification of PCR product for cap RNA synthesis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">dNTP mix</td>
			<td style="width:229px;">Promega</td>
			<td style="width:195px;">U1511</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tag DNA Polymerase</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:195px;">10342-053</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;"></td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;"></td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Equipment</td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">NanoDrop sepctrophotometer</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:195px;">ND-1000</td>
			<td>For measure morpholino and RNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Air Compressor</td>
			<td style="width:229px;">Werther International, Inc.</td>
			<td style="width:195px;">Panther Compact 106</td>
			<td>Air source for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Pico micro-injection pump</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">PV830 Pnematic PicoPump</td>
			<td>Other types of microinjection system can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Micro-manuplator</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">MMJR</td>
			<td>Right-handed (MMJL for left handed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Needle holder</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">5430-ALL</td>
			<td>To hold needle for micromanipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dumont Tweezers</td>
			<td style="width:229px;">Fine Surgical Tools</td>
			<td style="width:195px;">11253-20</td>
			<td>For breaking off the needle tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dissecting microscope</td>
			<td>Leica M205C&#160;</td>
			<td style="width:195px;"></td>
			<td>For observing and imaging zebrafish embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Fluorscence microscope</td>
			<td>Zeiss Axio Obserer D1m &#160;</td>
			<td style="width:195px;"></td>
			<td>For imaging zebrafish pronephros</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Capillary tube (I.D 0.15 mm)</td>
			<td>VitroCom</td>
			<td style="width:195px;">CV1525Q-100</td>
			<td>For measure the volume of each injected drop</td>
		</tr>
	</tbody>
</table>

a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

Wnt5a yıkmak çeviri MO engelleme getirerek elde edildi (Ağustos-MO) veya bir hücre aşamada Zebra balığı embriyolarının için ekson / intron sınır ekleme MO (ekleme-MO). Ağustos-MO nedenle, hem anne ve zigotik Wnt5a mesajını engeller, başlangıç ​​kodonu hedef ve. ek yeri MO, üçüncü parça verici bölge hedef ve Wnt5a (Şekil 1A) yalnızca zigotik transkript inhibe eder. AUG- ve splice- morphants kıvırcık kuyruk altındaki vücut ekseni ve kalp çevresi ödem...

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polikistik Böbrek Hastalığı Olası Zebra balığı Modeli: demonte<em&gt; Wnt5a</em&gt; Zebra balığı Böbrekler Kistler Nedenler

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

a-possible-zebrafish-model-polycystic-kidney-disease-knockdown-wnt5a

Research

JoVE Journal

Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

11.0K Görüntüleme.  Eastern Virginia Medical School. We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. 

Video: A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

Yetişkin Zebra balığı Myokard İnfarktüsü İndüksiyon Cryoinjury kullanma

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Tıp

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal Alkol Pozlama bir Zebra balığı Modelinde teratojenik değişiklikler değerlendirilmesi

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polikistik Böbrek Hastalığı Olan İnsanlar ve Fareler kromozom anomalileri Eğitim Spektral Karyotipik

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Bir Zebra balığı Otokton Tümör Modeli kullanılarak Melanom Değiştiricileri için Tarama

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes Mellitus ve Metabolik Belleğinin Zebrafish Modeli

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Kronik Böbrek Hastalarında Vasküler Fonksiyon Değerlendirilmesi

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Için Polikistik Böbrek Hastalığında Küçük Kemirgen modelleri Ultra yüksek Saha MRI Kullanımı<em&gt; In Vivo</em&gt; Fenotiplendirme ve İlaç İzleme

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Yetişkin zebra balığı doksorubisin kaynaklı kardiyomiyopati modelinde

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Fare modelleri Glomerular hastalığı böbrek fonksiyonlarının değerlendirilmesi

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...