Name: cryopreservation of zebrafish spermatogonia by whole testes needle immersed ultra-rapid cooling
Uploaded: 2018-03-04T13:00:00.000+00:00
Duration: 13 min 32 s
Description: Watch this Scientific Journal Video about Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling at JoVE.com

这项研究得到了匈牙利国家研究、发展和创新办公室 (赠款116912至ÁH)、成本办公室 (粮食和农业成本行动 FA1205: AQUAGAMETE)、Stipendium Hungaricum 奖学金方案 (赠款给各部) 和新匈牙利国家优秀 Predoctoral 奖学金 (授予克朗)。

这里描述的所有方法都已获得匈牙利动物福利法的批准。
1. 试剂制备
<ol><li>库存解决方案<ol>
<li>将1米海藻糖加入0.378 克海藻糖, 放入1毫升的 dH2o 混合均匀, 直到完全溶解。</li>
		<li>将1米蔗糖加入0.342 克蔗糖, 放入1毫升的 dH 中2o 混合均匀, 直至完全溶解。</li>
		<li>1米 HEPES 通过添加0.238 克 HEPES 到1毫升的 dH2o 混合好, 直到它完全溶解。</li>
		<li>通过增加100毫克到5毫升的 PBS, 制备20毫克/毫升胶原酶 (290 U/毫克)。彻底混合, 直到溶解。无菌过滤器通过0.2 µm 过滤器。整除50µL 到0.2 毫升 PCR 管和冷冻20摄氏度。</li>
		<li>通过增加75毫克到5毫升的 PBS, 制备15毫克/毫升胰蛋白酶 (~ 10000 U/毫克)。彻底混合, 直到溶解。无菌过滤器通过0.2 µm 过滤器。整除50µL 到0.2 毫升 PCR 管和冷冻20摄氏度。</li>
		<li>通过增加5毫克到5毫升的 PBS, 制备1毫克/毫升 DNase I (~ 3000 U/毫克)。彻底混合, 直到溶解。无菌过滤器通过0.2 µm 过滤器。整除50µL 到0.2 毫升 PCR 管和冷冻20摄氏度。</li>
		<li>加入40毫克的台盼蓝到10毫升的 PBS, 准备0.4% 台台盼蓝。无菌过滤器通过0.2 µm 过滤器。整除1毫升成1.5 毫升管。</li>
		<li>200毫克/升 MS-222 (Tricaine 甲烷磺酸), 加入200毫克的 MS-222 到1升的 dH2o 混合均匀, 直到它完全溶解。另外, 用碳酸氢钠将 MS-222 溶液缓冲到中性 pH 值为7.0。</li>
	</ol>
</li>
	<li>玻璃化解决方案<ol>
<li>准备平衡溶液: 通过混合121.5 µL 甲醇 (甲醇) 制备2毫升 ES; 1.5 米), 220 µL 丙二醇 (PG; 1.5 米), 200 µL 的血清 (10%), 200 µL 的海藻糖溶液 (0.1 米), 50 µL HEPES 库存溶液 (25 毫米) 和1208.5 µL L-15 成2 毫升管。</li>
		<li>准备玻璃化溶液 (vs): 准备2毫升的 VS 通过混合439µL 的 PG (3 米), 426 µL 二甲基亚砜 (i2因此; 3 M), 200 µL 的血清 (10%), 200 µL 海藻糖溶液 (0.1 M), 50 µL HEPES 库存溶液 (25 毫米) 和685µL L-15 成2毫升管。</li>
	</ol>
</li>
	<li>暖化解决方案<ol>
<li>准备变暖溶液 1 (WS1): WS1 2 毫升管中的1.5 毫升, 混合150µL 的血清 (10%), 450 µL 蔗糖溶液 (3 米) 和900µL L-15。</li>
		<li>准备变暖溶液 2 (WS2): WS2 2 毫升管中的1.5 毫升, 混合150µL 的血清 (10%), 150 µL 蔗糖溶液 (1 米) 和1200µL L-15。</li>
		<li>准备变暖溶液 3 (WS3): 通过混合150µL (10%) 和1350µL L-15, 在2毫升管中制备1.5 毫升 WS2。</li>
	</ol>
</li>
	<li>准备消化液: 为每个样品准备500µL 的消化液混合50µL 胶原酶的溶液 (2 毫克/毫升), 50 µL 胰蛋白酶溶液 (1.5 毫克/毫升), 10 µL 的 DNase I (20 µg/毫升) 和390µL L-15 在2毫升管。</li>
	<li>获得解剖工具: 剪刀, microscissors, 镊子, 弯镊子, 针灸针。</li>
</ol>2. 睾丸收集
<ol><li>弄死把鱼放进含有200毫克/升 MS-222 的盘子里 (pH = 7)。在继续解剖之前, 要确保鳃已经停止移动, 并且从那一刻起至少有10分钟通过, 以确保缺氧致死。</li>
	<li>用纸巾把鱼擦干, 把它放在解剖垫上的背侧。<ol>
<li>首先切除骨盆和胸鳍, 以便于解剖。</li>
		<li>水平剪下的鱼腹部的皮肤在胸鳍之间, 并削减皮肤和底层肌肉沿腹部, 直到肛门鳍。</li>
		<li>打开鱼的体腔, 将左、右体壁固定在带针的解剖垫上。 注: 睾丸位于游泳膀胱两侧的胃肠器官上方 (图 1)。</li>
	</ol>
</li>
	<li>为了防止污染, 不要取出胃肠器官, 而是轻轻地把它们推到一边, 从对方的睾丸中取出。睾丸被连接到身体壁上, 所以要小心地取出它们, 用 microscissors 切开连接的腹膜。</li>
	<li>此外, 为了防止污染, 首先对睾丸进行消毒, 将其放入70% 乙醇中2秒, 然后将它们放在冰上的 96 L-15 上。 注: 如有必要, 从脂肪组织和大血管中精确清除睾丸显微镜</li>
</ol>3. 睾丸组织的超快速冷却
<ol><li>根据样品类型, 用适当的标签标记针头, 并准备平衡 (ES) 和玻璃化溶液 (VS)。</li>
	<li>将睾丸转移到更大的井板或盘子上, 并将两到三个睾丸 (或更多取决于针的大小) 放在无菌针刺针上。<ol>
<li>首先将睾丸放在弯镊子的顶端。把针的点放在睾丸中间, 小心地用镊子把睾丸向上拉。</li>
		<li>刺穿睾丸后, 轻轻地将其向上拉向针的顶端。确保睾丸分离, 并没有脱落或下滑。</li>
		<li>将针与固定的睾丸放置在2毫升的 L-15 管中, 直到玻璃化 (25 ˚C; 贮存时间不应超过1小时)。 注: 将睾丸固定在针上应迅速进行, 以防止睾丸干燥。</li>
	</ol>
</li>
	<li>将针与睾丸转移到平衡溶液中, 在25˚C 孵育5分钟。</li>
	<li>将针头转移到玻璃化溶液中, 在25˚C 孵化三十年代。</li>
	<li>从玻璃化溶液中取出针头, 用无菌纸巾迅速地从组织中吸收剩余的溶液。小心, 避免睾丸粘在纸巾上或脱落。</li>
	<li>快速将针头放进发泡胶盒中的液氮中。 注意: 快速做到这一步, 以避免暴露组织到液氮蒸气。使用少量的液氮来防止样品的交叉污染。</li>
	<li>在封闭的发泡胶盒中, 将针放在液氮中 5-10 分钟。</li>
	<li>预冷打开了4.5 毫升 cryotube 和它的帽子在同一个盒子里。</li>
	<li>5-10 分钟后, 将每根针转移到液氮表面下的单独 cryotube, 并关闭 cryotube。 注意: 当接触液氮时, 经常使用防护服 (如绝缘手套), 因为暴露会导致严重冻伤。</li>
	<li>将 cryotube 放在金属手杖上, 并尽可能快地将其放在精子银行罐中。将样品储存在罐中储存的杜瓦瓶中, 直到进一步使用。</li>
</ol>4. 升温程序
<ol><li>每根针分别加热。所有变暖的解决方案应该在摄氏25摄氏度。</li>
	<li>将 cryotube 从金属藤中分离出来, 并将其浸入发泡胶盒中的液氮中。</li>
	<li>打开 cryotube 在液氮蒸气 (使用钳) 和释放针进入液氮。</li>
	<li>将针头快速转移到第一个变暖溶液中, 孵育1分钟 (摄氏25摄氏度)。一定要迅速转移针, 以防止其在空气中的变暖在转移。</li>
	<li>将针头转移到第二个变暖溶液中, 孵育3分钟 (摄氏25摄氏度)。</li>
	<li>将针头转移到第三种变暖溶液中, 孵育5分钟 (摄氏25摄氏度)。</li>
	<li>用弯曲的镊子向下滑动, 从针头中释放睾丸。将每个温暖的睾丸单独放置到一个单独的井 (96 井板块) 填充250µL L-15 补充10% 的血清。确保井被标记根据样品。把井板放在冰上, 直到所有样品都加热, 直到进一步工作。</li>
</ol>5. 组织消化
<ol><li>在分离的2毫升管中为每个样品准备500µL 的离解溶液。根据样品的代码给管子贴上标签。在室温下孵育5分钟制备的离解溶液。</li>
	<li>将温热的组织加入消化液中, 并将其切成小块, 至少30小剪刀的运动。</li>
	<li>在25˚C 的震动板上孵育90分钟的切口组织。</li>
	<li>通过添加400µL 的 L-15 和100µL 的血清 (10% 的血清 v/v) 来停止消化过程。在室温下, 简单地摇动溶液, 孵育1分钟。</li>
	<li>通过50µm 过滤器将获得的溶液过滤成1.5 毫升管。相应地给管子贴上标签。</li>
	<li>离心过滤溶液在200×g 10 分钟在25˚C。</li>
	<li>小心取出上清液和并用重悬在20µL 的 L15 补充10% 的血清。手动摇动管, 直到颗粒完全溶解。保持细胞悬浮在冰或4˚C, 直到进一步使用。</li>
</ol>6. 生存能力评估和细胞计数
<ol><li>将5µL 的细胞悬浮液和5µL 0.4% 台盼蓝溶液混合在适当标记的0.2 毫升 PCR 管中。如果使用不同的卷, 请确保1:1 的稀释率保持不变。</li>
	<li>在室温下孵化1至3分钟的悬浮液 (25 摄氏度)。</li>
	<li>检查显微镜下 hemocytometer 中每个样品的可行性。</li>
	<li>在15个字段中计算活细胞数 (无瑕由台盼蓝)。计算细胞悬浮液中20µm 的细胞总数。暂停现已准备好进行任何下游应用。 注意: 当对其他体型较大的物种进行优化时, 使用同样重量的睾丸碎片。为所有实验组使用睾丸, 以避免结果中的个体变异。在斑马鱼的情况下, 我们利用的事实, 左和右睾丸应包含相同 (或非常相似) 的生殖细胞数量13 (见代表性结果)。左睾丸保持作为一个控制, 而右睾丸是玻璃化/温暖。与新鲜睾丸分离出的细胞数量相比, 计算出的细胞数量与玻璃化/暖化睾丸分离的单元数相对应。</li>
</ol>

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作者没有什么可透露的。

本研究的主要目的是适应为禽类和哺乳动物物种开发的针浸入式超快速冷却程序10,11,12冷冻鱼睾丸 (斑马鱼作为模型有机体)。以往关于斑马鱼遗传资源超低温保存的研究主要集中在斑马鱼精子的超低温保存上2,3,4。然而, 成熟卵母细胞和胚胎的冷冻保存协议?...

科学和生物技术方面的新趋势导致创造了数以千计的新的突变体, 如小鼠、果蝇、斑马鱼和其他物种, 它们被用作生物医学和其他科学中的模范有机体1。此外, 随着新技术的开发和使用, 变异线的数量稳步增加2。这就导致了在保持繁殖殖民地的共同做法之外, 安全储存遗传资源的必要性。冷冻保存作为一种保证遗传资源在不确定时间内安全贮存的方法, 具有延长繁殖季节的优势, 绕过了对亲鱼持续维护的需要, 成本更高, 而且劳动效率2。
在过去几年中开发的精子超低温保存协议2,3,4为斑马鱼雄性遗传物质的成功储存提供了机会。然而, 由于其结构复杂, 蛋黄物质大量存在, 鱼的卵或胚胎的超低温保存还是不可能的。最近, 移植的原始生殖细胞 (PGCs) 或精原干细胞 (干细胞) 提供了一个旁路到这个障碍通过发展成功能精子和卵子移植后的5。因此, 干细胞的超低温保存为珍稀珍贵的遗传资源的保护提供了新的前沿。
尽管超低温保存提供了许多优点, 但慢速冻结过程会产生几种可能导致细胞损伤的条件2。其中包括细胞内和胞外冰的形成、脱水、cryoprotectant 毒性等。细胞内的冰会损伤癌细胞, 细胞外的冰可能会导致机械破碎, 而在慢速冷冻过程中细胞的水扩散可能导致脱水6。最近, 玻璃化作为一种防止冰形成的负面影响的技术已被应用于鱼类配子的超低温保存7,8,9。它提供了一种超快速冷却技术, 通过它, 内部和外部介质变成非晶态/玻型状态而不 crystalizing 到冰7,10。成功的玻璃化睾丸和卵巢组织已证明在鸟类和哺乳动物物种10,11,12, 从而打开它的应用在鱼的可能性, 以及。
在本研究中, 我们提出了针浸泡玻璃化的程序, 以保存整个斑马鱼睾丸。我们展示了一种可靠的方法, 以隔离斑马鱼的早期生殖细胞没有污染和冷冻过程, 产生相对较高数量的早期生殖细胞与低存在的其他细胞, 特别是精子。根据我们的知识, 这是第一项研究, 以演示一个详细的可视化协议, 以超快速冷却鱼类性腺组织和斑马鱼生殖细胞。此外, 该方法的重复性表现在五种不同的斑马鱼菌株: AB 野生型, 精灵 (罗伊-/;珍珠层-/), 豹 (leot1/t1), [tg (输精管:: eGFP)] 和肾母细胞肿瘤 [tg (wt1b::eGFP1)] 转基因线。

<table><tbody><tr><td>Leibovitz media (L-15)</td><td>Sigma-Aldrich</td><td>L1518</td><td>Supplemented with L-glutamine</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F9665</td><td></td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Sucrose</td><td>Acros Organics</td><td>57-50-1</td><td></td></tr><tr><td>Trehalose</td><td>Acros Organics</td><td>99-20-7</td><td>Dihydrate</td></tr><tr><td>Methanol</td><td>Reanal</td><td>20740-0-08-65</td><td></td></tr><tr><td>Propylene glycol</td><td>Reanal</td><td>08860-1-08-65</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Reanal</td><td>00190-1-01-65</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>9001-12-1</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8003</td><td></td></tr><tr><td>DNase I</td><td>Panreac AppliChem</td><td>A3778</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>T6146</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td>Tablets for preparation of 200 ml PBS solution</td></tr></tbody></table>

cryopreservation of zebrafish spermatogonia by whole testes needle immersed ultra-rapid cooling

目前科学和生物技术的趋势导致在模型生物体中创造数以千计的新品系, 从而导致有必要采用新的方法来安全地储存遗传资源, 使之超越饲养殖民地的共同做法。本研究的主要目的是适应针浸泡玻璃化 (cryopreserve) 程序, 以使整个斑马鱼睾丸。早期生殖细胞的全睾丸冷冻保存为斑马鱼遗传资源的贮藏提供了可能, 特别是在移植后它们可以成熟为雄性和雌性配子。睾丸被切除, 固定在针刺针上, 平衡在两个低温保护介质中 (含1.5 米甲醇和1.5 米丙烯乙二醇的平衡溶液; 含有3米二甲基亚砜和3米丙二醇的玻璃化溶液)并陷入液态氮。在一系列的三种升温溶液中, 样品得到了升温。这项技术的主要优点是 (1) 在热睾丸消化后缺乏精子, 从而促进下游操作;(2) 超快速冷却, 使组织的最佳接触液氮, 从而最大限度地冷却和减少所需的冷冻保护剂浓度, 从而减少其毒性;(3) 多睾丸同步暴露于冷冻保护剂和液氮;(4) 可重复性表现为在五种不同的斑马鱼菌株中获得50% 以上的生存能力。

平均来说, 从一个新鲜的斑马鱼睾丸中分离出来的早期生殖细胞的数量在4万到20万个细胞之间变化, 这取决于鱼的大小。在所有5株中消化新鲜的斑马鱼睾丸时, 早期生殖细胞并不是细胞悬浮液中唯一存在的细胞 (图 2)。在早期生殖细胞旁边, 也发现了许多精子。另一方面, 在冷冻后的睾丸中, 精子的消化少得多。这表明精子在超快速冷却协议中无法存活,...

Watch this Scientific Journal Video about Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling at JoVE.com

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

本研究的主要目的是适应针浸泡玻璃化 (cryopreserve) 程序, 以使整个斑马鱼睾丸。另外, 对五种不同斑马鱼菌株的重复性进行了测试。

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

cryopreservation-zebrafish-spermatogonia-whole-testes-needle-immersed

Research

JoVE Journal

Biology

8.3K 次观看.  Szent István University. 本研究的主要目的是适应针浸泡玻璃化 (cryopreserve) 程序, 以使整个斑马鱼睾丸。另外, 对五种不同斑马鱼菌株的重复性进行了测试。

视频: Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

科研

发育生物学

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

遗传学

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

筛选精子以快速分离斑马鱼的种系编辑

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

生物学

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

初压雌核发育二倍体的斑马鱼

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

<meta charset="utf8"></head><body>获得颗粒无细胞 I 期斑马鱼卵母细胞的改进程序

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.

<meta charset="utf8"></head><body>作者聚焦：分离纯斑马鱼 I 期卵母细胞的增强方法

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific research.

在无颗粒细胞的斑马鱼中快速分离 I 期卵母细胞

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

摘要

探索更多视频

此视频中的章节

斑马鱼胚胎的倍性操作与热休克治疗2

从斑马鱼精子细胞中培养染色体传播

筛选精子以快速分离斑马鱼的种系编辑

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和施肥

初压雌核发育二倍体的斑马鱼

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

在无颗粒细胞的斑马鱼中快速分离 I 期卵母细胞

在无颗粒细胞的斑马鱼中快速分离 I 期卵母细胞

在无颗粒细胞的斑马鱼中快速分离 I 期卵母细胞

生产单倍体斑马鱼的胚胎通过