作者感谢 IC 布鲁斯博士的批评性评论和阅读手稿。这项工作得到了中国国家自然科学基金 (31430059、31701272、31730061、81470399和 31521062)、亚洲经济和新兴市场创新医学和早期发展的资助。

所有动物程序都使用了由北京大学机构动物护理和使用委员会批准的斑马鱼议定书, 该协议由实验室动物护理评估和认可协会完全认可。
1. Tricaine 溶液的制备
<ol><li>要准备 tricaine 的溶液, 添加400毫克 3-氨基苯甲酸甲基磺酸粉到97.9 毫升蒸馏水, 然后加入2.1 毫升的1米 (ph 9.5) 调整 ph 值 ~ 7。将库存解决方案存储在摄氏4摄氏度。</li>
	<li>为准备 tricaine 工作解决方案, 添加4.2 毫升 tricaine 库存解决方案, 100 毫升水族系统水。</li>
</ol>2. 成年斑马鱼心室切除术
<ol><li>为手术准备器械和材料, 包括一块海绵, 培养皿中充满 tricaine 工作溶液, 塑料吸管, 显微镜, 两个锋利的镊子, 虹膜剪刀和肘钳 (图 1)。</li>
	<li>在海绵中准备一个直径为4厘米 x 0.5 厘米和0.5 厘米的小凹槽, 在心室切除手术中保存斑马鱼。</li>
	<li>用60毫克/升海水盐在弶镀 ' p2O 为受伤的鱼回收, 在3升斑马鱼罐内加装半罐斑马鱼系统水。</li>
	<li>麻醉鱼在一个10厘米培养皿与 tricaine 溶液, 直到鳃运动减少到每秒, 并保持鱼至少三十年代, 以确保它完全麻醉。</li>
	<li>将麻醉鱼与肘部镊子转移到湿海绵腹侧的凹槽上。视觉定位跳动的心脏的边缘, 然后找到显微镜下的心脏。</li>
	<li>使用虹膜剪刀做一个小切口约1毫米, 穿透皮肤和肌肉, 并仔细撕裂的银色上皮层的心包囊与镊子提示进入心脏。不要把镊子插入太深的腔内以避免心脏损伤。 注意: 在这一点上, 请记住避免大量出血。排除鱼的实验, 如果广泛出血发生, 因为过量出血主要是由于意外伤害心脏, 因此将导致误导的结果。</li>
	<li>使用非显性的手或施加温和的腹部压力, 用锋利的镊子轻轻地握住它的顶端以暴露心室。然后, 用虹膜剪刀把20% 的心室放在顶端。 注: 此时不应发生广泛出血。</li>
	<li>用湿纸巾覆盖切口, 因为伤口在凝固前 15–45, 然后用系统水把鱼放回水箱里。 注意: 没有必要缝合切口。</li>
	<li>确保鱼在1分钟内恢复鳃运动, 然后继续游泳。如果鱼在五十年代以后不显示鳃运动在水中, 促进它通过喷水入鳃使用塑料吸管, 直到鱼恢复活跃透气。</li>
	<li>在心室切除后的回收系统中保持鱼。 注意: 与普通鱼相比, 没有必要特别关心。成年斑马鱼心室切除术的死亡率约为5–10%。几乎所有的死亡发生在手术后24小时内。</li>
</ol>3. siRNA 纳米粒子的制备
<ol><li>合成了 f-PAM4-PEG-R9 大分子。 注: 对 f-PAM4-PEG-R9 大分子材料的合成进行了前述37的一些修改。α、ω-dipyridyl disulfido 聚乙二醇 () 的长度降低为分子量的 1000, 以最大程度地封装 siRNAs (图 2B)。</li>
	<li>干10毫克的半胱胺核心 G4.0 polyamidoamine (称为 PAMAM) 使用真空干燥器在45摄氏度1小时, 然后溶解 PAMAM 在2毫升 Dulbecco 的磷酸盐缓冲盐水 (DPBS, 平衡盐溶液用于各种细胞培养应用)。</li>
	<li>将1.7 毫克的三 (2-羧乙基) 膦 (TCEP) 溶于1毫升 DPBS, 与3.2 制备的 PAMAM 溶液混合, 在室温下以温和搅拌为 8 h。 注: TCEP 的摩尔比超过 PAMAM 的二硫化物键的十倍。</li>
	<li>通过超滤去除过量 TCEP。添加 DPBS 到上述 PAMAM-TCEP 混合物达到10毫升的体积和解决方案转移到 3 kDa 截留分子量 (分子量切断) 超滤管。离心后以3000克为15分钟, 在收集管中丢弃溶液。将 DPBS 添加到10毫升的过滤器中, 并轻轻地将样品多次吸出并再次离心。重复此步骤4次。最后一次过滤后, 从过滤器中抽取样品。 注: 此步骤的产品减少 PAMAM (称为 f-PAM4)。</li>
	<li>在1毫升 DPBS 中溶解14毫克的 PEG, 并与 f-PAM4 溶液混合, 在室温下保持3小时的温和搅拌。然后用3.4 中描述的超滤步骤去除过量的脱钉。最后一次过滤后, 从过滤器中抽取样品。 注: 该产品被命名为 f-PAM4-PEG-Py。f-PAM4 中的硫醇组的摩尔比值为10倍。</li>
	<li>将6.4 毫克半胱氨酸终止 R9 肽 (Ac-CRRRRRRRRR-NH2) 溶于1毫升 DPBS, 与 f-PAM4-PEG-Py 混合, 在室温下轻轻搅拌8小时。净化最后的产品, 称为 f-PAM4-PEG-R9, 由超滤步骤, 如3.4 所述, 除了 DPBS 被改为去离子水。使用冷冻真空干燥机 Lyophilize 产品。 注: 冻干前3.5 和3.6 的产品储存在-20 ℃至少1月。在 f-PAM4-PEG-R9 冻干后, 样品储存在-20 ℃, 至少2月。</li>
	<li>为了确定 PEG 和肽的改性程度, 将5毫克的 f-PAM4-PEG-Py 在 D2O (氘氧化物) 中溶解, 并将其转移到 NMR (核磁共振) 样品管中。在 400 MHz 进行1的 h-核磁共振谱分析, 并用核磁共振数据分析软件37分析数据。 注: 具体而言, 多肽中的代表性 PAMAM 峰 (-NCH2CH2CO)、PEG 峰 (-OCH2CH2) 和精氨酸残留峰 (-HCCH2CH2CH2NH), 应分别在2.4–2.6、3.7–3.8 和 1.6–1.8 ppm 范围内。通过计算 peg 和 R9 峰到 PAMAM 峰的积分, 确定 R9 相对于 PAMAM 的改性程度。PEG 的改性程度为 100%, R9 改性至少为50%。在该机构的核心设施中进行了1的 H 核磁共振谱分析。</li>
	<li>将4-PEG-R9 大分子粉在 PBS 中溶解至4毫克/毫升, 并使整除数溶液一次性使用。 注: 大分子溶液可储存在-20 摄氏度, 至少一个月, 不重复冻结和解冻。</li>
	<li>将 siRNAs 中的菁染料 (称为 Cy5-siRNA)、Aldh1a2 siRNA 或炒阴控制 siRNA 在蒸馏水中溶解至10µM。 注: 使用菁染料标记的 siRNAs, 以评估纳米颗粒封装的 siRNA 是否可以进入鱼心 (图 2A B)。该协议以aldh1a2为例, 确定 f-PAM4-PEG-R9 大分子封装 siRNA 的传递效率。siRNAs 的序列为: Cy5-siRNA 反义链: 5′-ACGUGACACGUUCGGAGAAdTdT 3′, siAldh1a2 反义链: 5 '-UUCAGGACAACCGUGUUCCdTdT-3 ' 和炒负控制 siRNA 反义链: 5 '-ACGUGACACGUUCGGAGAAdTdT-3 '。</li>
	<li>为制备 siRNA 封装的纳米微粒溶液, 将 siRNA 与大分子溶液的等量量混合, 并在室温下孵化样品20分钟. 在使用前新鲜地准备 siRNA 纳米微粒溶液。 注: siRNA 纳米粒子溶液的体积取决于要注射的鱼的数量。例如, 准备至少120µL 大分子溶液 (混合60µL siRNA 溶液和60µL 大分子溶液), 用于注射10条鱼 (每条鱼的10µL), 计算出溶液的损失。</li>
</ol>4. 将 siRNA 纳米微粒注射入成年斑马鱼心脏
<ol><li>如上所述, 准备一块海绵与槽, 一个10厘米培养皿填充 tricaine 溶液, 一个塑料吸管, 显微镜, 肘部镊子, 和胰岛素注射器 (图 1), 以及一个鱼缸与系统水的住房鱼注射后。</li>
	<li>允许受伤的斑马鱼2.1–2.10 在心室切除术后1天恢复, 然后麻醉 1 dpa (在截肢后的几天内) 鱼, 如2.4 所述, tricaine 溶液中。</li>
	<li>用肘钳将麻醉鱼、腹侧向上转移到湿润海绵中的沟槽。在显微镜下找到跳动的心脏。</li>
	<li>将 siRNA 封装的纳米微粒溶液加载10µL 胰岛素注射器。尽量避免气泡。</li>
	<li>用肘钳轻轻地将鱼固定在不占主导地位的手上。不要把鱼夹得太紧。 注: 请记住, 注射过程中不应发生广泛出血。</li>
	<li>将填充的胰岛素注射器放在µL, 用另一只手将10的溶液注入胸腔 (补充图 1)。 注: 溶液有时在注射过程中流出, 但这并不影响 siRNA 效应。siRNA 纳米微粒溶液可以每天注射一次, 为期数天到一个月, 这取决于具体的实验计划。每次给鱼注射一种 siRNA。如果有两种以上的 siRNA 需要注入一条鱼, 每次注射一种 siRNA, 然后再注入另一种类型的 siRNA 1 小时。在本议定书中, 每条鱼只注射一种 siRNA 类型。</li>
	<li>用系统水把鱼转移到水箱里。 注: 该鱼有望在1分钟内重新启动通风, 然后恢复游泳。注射 siRNA 纳米颗粒或 siRNA 通常不会导致成年斑马鱼死亡。</li>
</ol>5. 心脏的收集、固定和 siRNA 分娩效率的评价
<ol><li>在离心管中为每个组准备1毫升的4% 多聚甲醛溶液。</li>
	<li>麻醉在 tricaine 溶液中的鱼在注射后选定的日子。将鱼仰在潮湿的海绵中放入凹槽中。在胸腔内做一个大切口, 用镊子广泛开放。抓住流出道, 用镊子小心翼翼地拔出整个心脏。</li>
	<li>用 PBS 冲洗心脏, 用餐巾快速取出多余的液体。将10颗心放入1.5 毫升的离心管中, 1 毫升多聚甲醛。轻轻地将管子反转几次, 并保持隔夜在4摄氏度。</li>
	<li>使用体内成像系统测量 Cy5-siRNAs-injected 心脏的荧光强度 (补充图 2)。在这项研究中, 每组使用3–4斑马鱼的心脏。</li>
	<li>安装固定的心脏, 无论是石蜡或 OCT 化合物 (嵌入培养基为冷冻组织标本, 以确保最佳切削温度)。</li>
</ol>6. 纳米粒子介导的 siRNA 基因沉默的评价
<ol><li>准备一个隔热容器和长镊子。将液氮倒入容器容积的第三位。</li>
	<li>麻醉鱼在 tricaine 解决方案在 2 dpa。解剖心脏如5.2 所述, 然后去除流出道和心房。 注: 鱼可以在一天内从手术中恢复, 然后注入 1 dpa, 其中任一 siRNA 封装纳米微粒 (siNC) 为阴性对照或 aldh1a2 siRNA 封装纳米粒子 (siAldh1a2)。</li>
	<li>使用镊子将心室转成1.5 毫升的离心管, 盖上管盖, 并立即将管子放入带有液氮的绝缘容器中。让管子在液氮上漂浮几分钟. 使用长镊子从容器中取出管子。</li>
	<li>重复步骤6.2 和 6.3, 直到所有心室被收集。每组使用6–10心。</li>
	<li>用 rna 分离试剂提取心脏总 rna。用引物 Gapdh F: GATACACGGAGCACCAGGTT 对各基因的表达水平进行定量 rt-pcr 评价;Gapdh R: GCCATCAGGTCACATACACG;Aldh1a2 F: TGAGCGAGGAGCAGCAGAGA;Aldh1a2 R: TCCACGAAGAAGCCTTTAGTAGCA。</li>
</ol>

作者没有什么可透露的。

斑马鱼完全有能力再生各种器官, 包括成人心脏5。尽管转基因和遗传方法在研究斑马鱼胚胎中的基因功能方面发展良好, 研究者仍面临着在斑马鱼45,46中产生条件突变基因的艰巨任务。因此, 转基因显性阴性突变体或小分子抑制剂经常被用来处理成年斑马鱼器官的基因功能25,27,<sup class...

心肌梗死已成为严重的健康威胁, 导致1世界各地的巨大经济负担。成年哺乳动物心脏在损伤后无法再生和补充失去的心肌细胞, 导致瘢痕组织的形成和随后的心力衰竭。与哺乳动物不同的是, 斑马鱼能够心脏再生, 主要是通过各种类型的心脏损伤后的强健心肌增生, 使其成为研究心脏再生分子机制的理想模型有机体。2,3,4,5,6,7,8。破译斑马鱼心脏再生的内源机制是一个令人兴奋的研究领域, 寻找新的治疗策略, 以改善人类心脏再生9。
在斑马鱼身上有遗传操作方法。这些包括 morpholinos (MO), 也广泛用于青蛙, 小鸡和哺乳动物除斑马鱼10,11,12,13。MO 在成年斑马鱼鳍, 脑, 视网膜14,15,16,17,18,19, 有效地击倒靶基因表达。锁定核酸 (低噪声放大器) 是另一种用于除斑马鱼胚胎中的内源基因表达的人工寡核苷酸, 在成年动物器官中也有20,21,22,23,24. 然而, 对于成年人心脏缺乏有效的功能丧失方法, 仍然是研究器官再生分子机制的一个障碍。目前, 小分子抑制剂或显性阴性突变体的转基因表达主要用于阻断某一基因或通路的功能, 以研究其在斑马鱼心脏再生25、26 中的作用. ,27。然而, 并非所有的基因或信号通路都适用于这些方法。
小干扰 rna (siRNAs) 广泛应用于哺乳动物细胞和模型生物体胚胎的功能损失分析, 以及动物模型28、29、30的临床前研究的成人器官。,31,32. siRNAs 已有效地用于沉默的肿瘤33,34,35和心肌细胞36,37,38,39 的基因 ,40 通过不同的交付系统。最近, 我们开发了有效的 siRNA 封装纳米颗粒基因沉默的再生成人心脏使用几种不同的纳米粒子41,42,43, 提供了一个新的工具成年斑马鱼器官基因的功能研究。根据我们以前的研究41,42,43, 这里我们提出了一个简单的, 实用的, 但强大的协议 siRNA 基因沉默在再生成年斑马鱼心脏使用 f-PAMAM-PEG-R9大分子.Aldh1a2(醛脱氢酶1家族, 成员 A2) 基因上调后, 斑马鱼的先端切除和消融Aldh1a2阻断心脏再生44。以aldh1a2基因为例, 对纳米粒子包覆 siRNA 注射液介导的基因击倒效率进行了测试。本协议包含了斑马鱼心脏切除、纳米微粒的化学合成和 siRNA 包裹纳米微粒向成年斑马鱼心脏的传递方法。

<table><tbody><tr><td>tricaine</td><td>Sigma</td><td>E10521</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>stereomicroscope</td><td>Leica&amp;nbsp;</td><td>S8AP0</td><td></td></tr><tr><td>sharp forcep</td><td>WPI</td><td>14098</td><td></td></tr><tr><td>iridectomy scissors</td><td>WPI</td><td>501778</td><td></td></tr><tr><td>elbow tweezers</td><td>Suzhou Liuliu</td><td>SE05Cr</td><td></td></tr><tr><td>&amp;alpha;,&amp;omega;-dipyridyl disulfido polyethylene glycol(Py-PEG-Py)</td><td>Biomatrik (Jiaxing) Inc.</td><td>5239</td><td></td></tr><tr><td>core of G4.0 polyamidoamine (PAMAM)</td><td>Andrews ChemServices</td><td>AuCS-297</td><td></td></tr><tr><td>vacuum drying equipment</td><td>Yiheng</td><td>DZF-6020</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>tris(2-carboxyethyl)phosphine(TCEP)</td><td>Alfar Aesar</td><td>51805-45-9</td><td>Causes severe skin burns and eye damage. Causes serious eye damage.</td></tr><tr><td>ultrafiltration tube</td><td>Millipore</td><td>UFC900308</td><td></td></tr><tr><td>freeze dryer</td><td>Martin Christ</td><td>Alpha 2-4 Ldplus</td><td></td></tr><tr><td>NMR spectrometer</td><td>Bruker</td><td>AV400</td><td></td></tr><tr><td>Deuterium oxide(D&lt;sub&gt;2&lt;/sub&gt;O)</td><td>J&amp;amp;K</td><td>174611</td><td></td></tr><tr><td>NMR sample tube</td><td>J&amp;amp;K</td><td>WG-1000-7-50</td><td></td></tr><tr><td>3 kDa MWCO ultrafiltration tube</td><td>Merck</td><td>UFC900308</td><td></td></tr><tr><td>sea salts</td><td>Instant Ocean&amp;reg;</td><td>SS15-10</td><td></td></tr></tbody></table>

nanoparticle-mediated sirna gene-silencing in adult zebrafish heart

哺乳动物在心肌梗死后再生心脏的能力非常有限。另一方面, 成年斑马鱼在先端切除或 cryoinjury 后再生心脏, 使其成为心脏再生研究的重要模型机体。然而, 缺乏功能丧失的成人器官的方法, 限制了对心脏再生机制的洞察力。RNA 干扰通过不同的运载系统是一个强有力的工具为沉默基因在哺乳动物细胞和模型有机体。我们以前曾报道, siRNA 纳米粒子成功地进入细胞, 并导致在再生的成年斑马鱼心脏显着基因特定的击倒。在这里, 我们提出了一个简单, 快速, 有效的协议大分子介导的 siRNA 交付和基因沉默的再生成年斑马鱼心脏。该方法为确定斑马鱼成年器官中的基因功能提供了一种替代方法, 可推广到其他模型生物中。

为了确定大分子介导的 siRNA 分娩的效率, 我们切除了斑马鱼心脏心室的顶端, 然后注射了约10µL 的大分子 (模拟组), Cy5-siRNA 只 (裸组), 或 f-PAMAM-PEG-R9 大分子封装Cy5-siRNA (Cy5-siRNA 组) intrapleurally 分别 (图 2A B)。荧光信号是可检测的心脏注射大分子封装 Cy5-siRNA 在 3, 24 和 48 hpi (小时后注射), 而它是很难检测到心脏从模拟和裸组在 48 hpi (<strong cla...

Watch this Scientific Journal Video about Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart at JoVE.com

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

在成年斑马鱼器官中发展有条件基因剔除或有效基因击倒仍是一项重大挑战。在这里, 我们报告了在成年斑马鱼心脏执行纳米粒子介导的 siRNA 基因沉默的协议, 从而为研究斑马鱼和其他模型生物体中的成年器官提供了一种新的功能损失方法。

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

nanoparticle-mediated-sirna-gene-silencing-in-adult-zebrafish-heart

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Peking University. 在成年斑马鱼器官中发展有条件基因剔除或有效基因击倒仍是一项重大挑战。在这里, 我们报告了在成年斑马鱼心脏执行纳米粒子介导的 siRNA 基因沉默的协议, 从而为研究斑马鱼和其他模型生物体中的成年器官提供了一种新的功能损失方法。

视频: Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

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 ...

医学

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

神经科学

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

高频超声超声心动图评估斑马鱼心脏功能

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

<meta charset="utf8"></head><body>设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

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.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

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

生物学

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

摘要

探索更多视频

大规模的斑马鱼胚胎的心脏解剖的转录分析

在成年斑马鱼心肌梗死的感应使用Cryoinjury

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

高频超声超声心动图评估斑马鱼心脏功能

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

阿霉素诱发成年斑马鱼的心肌病模型

恢复成年斑马鱼的心脏的高通量应用

胸腔内注射治疗成年斑马鱼心脏的研究