这项工作是支持由美国国立卫生研究院（DK093625到LH和DK069909和DK047757至JHL）和VA（优异奖I01BX000820到JHL）。我们要感谢博士迈克尔·包和何洁博士在宾夕法尼亚斑马鱼核心的大学提供必要的支持。

注：伦理学声明：所有斑马鱼实验批准的机构动物护理和使用委员会在东弗吉尼亚医学院。 1.准备吗啉<ol><li>设计和合成的翻译阻滞（AUG-）和剪接抑制（剪接）反义吗啉代（MO）的寡核苷酸用于感兴趣按照制造商的说明（ 图1A）的基因。请参阅生产商的表1中的信息。 注：材料订单出货冻干股玻璃瓶。 </li><li>添加高档无菌水至玻璃瓶重新暂停的MO至25微克/微升的最终浓度。确保该寡核苷酸被完全溶解。如果一些固体遗骸，加热包含在65℃的股票的寡核苷酸溶液中5至10分钟，涡旋简要小瓶。 </li><li>存储在MO原液在RT。不要将它们存储在4℃或-20℃; C，因为较低的温度可能导致在MO寡核苷酸结合到容器壁。测量使用分光光度计原液的浓度（请参阅表1）一个新的MO原液制成各一次。 </li><li>通过稀释原液用高档无菌水至所需的剂量准备注射的当天将MO工作溶液。加0.5％酚红达到0.05％的浓度。例如，以与15毫微克/ nL的浓度5μl的工作溶液中，加入3微升的MO原液和0.5微升0.5％酚红至1.5微升水。 注：有了这个工作浓度，注射每一滴（500 PL）包含MO的7.5纳克和两滴含有15纳克MO的。 </li></ol> 2.准备的注射装置的<ol><li>购买玻璃预拉针（请见表1详细信息）。另外，拉玻璃注射针无线TH针拉马。 </li><li>将空气压缩机和调整压力设置为50磅。打开解剖显微镜光源和微微微量注射泵和调整设置如下：保持压力20磅，喷射压力10磅，2.5和100微秒范围选通的周期值。 </li><li>加载玻璃预拉针以5微升注射溶液和放置针在与尖端朝下的垂直位置。等待，直到所有的溶液到达针尖没有可见气泡。将针保持器在旁边的显微镜适当的位置，并插入玻璃针插入支架。调整喷射角为45度。 </li><li>把针尖进入视野，在显微镜下，高走下舞台，并专注于尖端最薄的区域。断针尖与细点镊子。 </li><li>将尺子和毛细管具有0.15mm的一侧通过显微镜下侧内径;该溶液注入到毛细管的一端，以使一个液柱。调整排出时间来达到每17滴每1mm长度的液柱，然后每滴的体积为1 NL（墨滴体积液柱=πX半径2×长度（）滴/计数（）。 注：将一滴用直径为0.1毫米给出约500复一个注射量（墨滴体积= 4/3 XπX半径3）。 </li></ol> 3.准备施肥斑马鱼胚胎和注射用吗啉代<ol><li>根据标准斑马鱼的饲养和维护协议公布转基因斑马鱼繁殖对：设置的Tg（GFP wt1b）。收集自然产卵后的胚胎，并将其保存在一个10cm培养皿充满E3水（5毫米氯化钠，氯化钾0.17毫米，0.33毫米氯化钙2，0.33毫米硫酸镁4）。启动MO注射马上13。监测胚胎形态，在显微镜下，并确保MO注射是在单细胞阶段，或不迟于四细胞阶段。 <ol><li>胚胎转移到10cm的培养皿中以楔形琼脂槽。吸出多余的水E3并轻轻按压胚胎进入低谷。 </li><li>操纵与微量的胚胎以可视化的解剖显微镜下1-细胞期的胚胎。穿透绒毛膜，然后将蛋黄到一个或两滴MO的注入蛋黄（1滴= 500 PL）。转移注射的胚胎至10cm的培养皿与E3的水和孵化在28.5℃。 </li></ol></li><li>在注射后，取出死胚，并记录注射的胚胎的数量。取代E3水在盘每24至48小时。 </li></ol> 4.表型救援实验<ol><li>选择来自另一个物种具有不同的主碱基对结构（通常为3-7个碱基对的错配），并且，因此，耐对MO的直向同源物，救援。例如，在这个实验中，我们选择了鼠标因为鼠标Wnt5a的 mRNA序列是来自斑马鱼的序列不同。 </li><li>设计的引物组与正向引物起始于平移位点和反向引物在接近所述mRNA编码区的末端。添加T7启动子序列的正向引物序列的5&#39;端。在这个实验中，我们使用了以下的引物序列;前锋：5&#39;-TAA TAC GAC TCA CTA标签GGA CTA TGA TGC TGC TGA AGC TGA A- 3&#39;和反向：5&#39;-TCA CTT GCA GAC GTA CTG gtc- 3“。 </li><li>执行一个50μl的聚合酶链反应（PCR）用引物组（0.5μM，每种引物）上述设计和0.1微克含有小鼠Wnt5a的 cDNA序列与下列PCR程序模板DNA：1个循环的95℃5分钟; 35个循环的95℃（30秒），58.5℃（30秒），72℃（1分钟）;和最后延伸步骤在72℃进行7分钟。 </li><li>完成后，吨他的周期，运行2微升的PCR产物的1％琼脂糖凝胶，以确保乐队是正确的大小。纯化的PCR产物与根据制造商的说明一PCR纯化试剂盒。检查DNA浓度用分光光度计。存储所述纯化的PCR产物，在-20℃或在加帽的RNA转录在下一步直接使用。 </li><li>合成体外根据制造商的说明书加帽的mRNA用加帽的RNA合成试剂盒。稀释的mRNA在20μl无RNA酶的水，并确定该浓度。分装的RNA，并储存于-80℃。 </li><li>对注射的当天，制备mRNA的核糖核酸酶与游离的无菌水的工作溶液。需要注意的是40皮克通常是在该RNA的非特异性或毒性作用不发生的最高的RNA剂量。置于冰上的工作方案。注入胚胎与MO，然后抢救的mRNA，或二者共同注入在一起。例如，如果与mRNA prepar的浓度每次注射的ED在步骤4.5为0.6微克/微升，添加0.67微升的mRNA（0.4微克）与4.33微升无RNA酶的水，使0.08微克/微升的最终浓度，然后一滴（500 PL）中含有40皮克mRNA的。准备MO如步骤3中所述。 </li></ol> 5.准备注射胚胎的荧光成像<ol><li>孵育28.5°CO后/ N，增加0.003％N-苯基硫氧嘧啶（PTU）的E3水，以防止黑化，从而影响观察用荧光显微镜前肾结构。然而，原肠胚形成之前不要添加PTU，因为它会影响早期胚胎发育。 </li><li>在48 HPF，手动dechorionate用细镊子胚胎。采取48和72 HPF胚胎图像光解剖显微镜下。 注意：这些图片可以采取无需麻醉。 </li><li>约10分钟前在荧光显微镜下成像，将它们放置在麻醉胚胎10cm的培养皿含有160微克/毫升缓冲三卡因。等待5-10分钟，然后轻轻地接触斑马鱼，以确认它们不移动，因此，麻醉正在工作。 </li><li>之后将胚麻醉，将它们安装在3％的甲基纤维素和定向它们在解剖显微镜下俯卧位置（请参阅表1）。 </li><li>观察在荧光显微镜下的前肾结构（请参阅表1），并拍摄照片，在不同的放大倍数（10X和20X）。 </li></ol>

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The authors have nothing to disclose.

多囊肾（PKD）是终末期肾脏疾病的主要原因在人类中之一，其特征在于进行性囊肿形成，肾肿大，与异常小管发育14。常染色体显性多囊肾（ADPKD）是一种遗传性疾病，其中突变要么PKD1的，编码多囊蛋白1（PC1），或PKD2，多囊编码-2（PC2），导致多囊性肾脏。许多其它基因，特别是那些编码在初级纤毛中发现的蛋白质，被认为是参与肾囊肿的发展，至今尚无理想的工具来筛选这些基因。我?...

斑马鱼（ 斑马鱼 ）胚胎已被广泛用作模型为研究肾发展和多囊肾病。有许多优势，使用斑马鱼作为动物模型：研究遗传相互作用的可行性，使用反义吗啉（MO），用于蛋白质敲低的能力，有机会迅速测定大量的胚胎，并且容易观察器官的表型活幼虫1。该前肾是第一个肾在脊椎动物发展和功能是在幼虫斑马鱼2。斑马鱼前肾的结构相对简单相比哺乳动物后肾，第三和最后肾发展的哺乳动物。肾单位是肾脏的工作单元，其中每个人肾脏500,000-1,000,000肾3,4和具有约13000肾5各小鼠肾脏之间含有，因此很难观察单肾结构我ñ人或小鼠肾脏。斑马鱼只有两个肾单位，每个斑马鱼肾包含所有在肾小球和小鼠和人类6和类似专门肾细胞类型的小管中发现的主要组件。相比其他脊椎动物模型如爪蟾 ，斑马鱼肾更接近于哺乳动物肾，因为它有一个封闭的系统7。 在最近几年中，斑马鱼的基因组已经被测序，允许广泛介绍的遗传工具，广泛突变体资源，并在斑马鱼的模型转基因报告线路集合。 12-72小时受精后（HPF）之间的斑马鱼前肾表格可在透明的胚胎很容易被可视化。在肾母细胞瘤蛋白WT1是肾脏发育的一个重要因素。所述wt1b启动子的Tg的控制下表达绿色荧光蛋白（GFP）转基因斑马鱼线（wt1b：GFP）显示GFP expre裂变专门设在pronephric地区的斑马鱼胚胎，从17 HPF 8起。 Nephronophthisis（NPHP），一种常染色体隐性囊性肾脏疾病，是由NPHP基因9的突变引起。 NPHP4被击倒吗啉导致囊肿形成的的Tg（wt1b：GFP）鱼10因此，这种转基因鱼是一个合适的模型，观察肾脏结构和囊肿形成肾脏发育过程中。重要的是，肾脏发育的调节剂的影响，可以利用该菌株中一个时间和劳动高效方式进行研究。 我们的论文描述了使用的Tg（wt1b：GFP）鱼作为模式基因调制后显现肾囊肿形成。我们使用的开始和剪切位点反义的MO打掉了Wnt5a基因斑马鱼。而Wnt5a是Wnt信号家族的非规范的分泌糖蛋白，发挥各器官和细胞产后的发展具有重要作用功能11。 Wnt5a的工作原理是通过非经典Wnt途径，包括平面细胞极性（PCP）途径，已经发现肾小管伸长时发挥导向细胞分裂的作用。 Wnt5a的通过形成与受体样酪氨酸激酶（RYK），其中，还转导Wnt5a的信令通过形成与VANGL平面细胞极性蛋白2（Vangl2）的复合物，从而促进Vangl2稳定性12复杂调节的Wnt / PCP通路。在PCP途径缺陷可能导致随机的细胞分裂，并导致肾囊肿形成。我们使用的Tg（wt1b：GFP）斑马鱼线以下的Wnt5a敲除，观察肾囊肿形成。 的Tg（wt1b：GFP）斑马鱼模型允许实时成像，及时观察肾脏结构。 Wnt5a的击倒后，肾囊肿形成被发现开始，在24 HPF;在72 HPF，囊肿可能在肾小球和近端小管中找到。这种方法也可用于为screen其他基因可能导致肾囊肿形成。

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	<tbody>
		<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>
		</tr>
		<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的击倒被引进翻译阻挡MO实现（AUG-MO）或外显子/内含子的剪接边界MO（拼接-MO），以斑马鱼胚胎的一个细胞阶段。所述AUG-MO靶向起始密码子，因此，抑制了母亲和合子Wnt5a的消息。拼接-MO靶向第三剪接供体位点和抑制Wnt5a的（ 图1A）仅合子转录。所述AUG-和剪接morphants phenocopied彼此具有多个缺陷，包括卷曲的尾巴向下体轴和心包水肿（ 图1B）。鼠标Wnt5a的mRN...

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.

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

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 次观看.  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. 

视频: 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.

在成年斑马鱼心肌梗死的感应使用Cryoinjury

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

科研

医学

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.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

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.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

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.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

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.

斑马鱼模型的糖尿病和代谢记忆

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.

血管功能在慢性肾脏疾病的评估

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.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

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.

阿霉素诱发成年斑马鱼的心肌病模型

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.

小鼠肾小球疾病模型肾脏功能的评价

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.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

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