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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Comp. Endocrinol. 245, 77-83 (2017).</li><li>P&#353;eni&#269;ka, M., Saito, T., Rodina, M., Dzyuba, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+early+stage+Siberian+sturgeon+Acipenser+baerii+germ+cells,+comparison+of+whole+tissue+and+dissociated+cells.">Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells.</a> Cryobiology. 72, 119-122 (2016).</li><li>Lee, S., Iwasaki, Y., Shikina, S., Yoshizaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eggs+and+sperm+from+cryopreserved+whole+testes.">Generation of functional eggs and sperm from cryopreserved whole testes.</a> Proc. Nati. Acad. Sci. USA. 110 (5), 1640-1645 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">Leibovitz media (L-15)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">L1518</td>
			<td style="width:296px;">Supplemented with L-glutamine</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:232px;">Fetal bovine serum (FBS)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">F9665</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine methanesulfonate (MS-222)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td>E10521</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:232px;">HEPES</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">H3375</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Sucrose</td>
			<td style="width:220px;">Acros Organics</td>
			<td>57-50-1</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trehalose</td>
			<td style="width:220px;">Acros Organics</td>
			<td>99-20-7</td>
			<td style="width:296px;">Dihydrate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Methanol</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">20740-0-08-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Propylene glycol</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">08860-1-08-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Dimethyl sulfoxide</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">00190-1-01-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Collagenase</td>
			<td style="width:220px;">Gibco</td>
			<td style="width:200px;">9001-12-1</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trypsin</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8003</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">DNase I</td>
			<td style="width:220px;">Panreac AppliChem</td>
			<td style="width:200px;">A3778</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trypan blue</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">T6146</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Phospate buffered saline (PBS)</td>
			<td style="width:220px;">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

8.2K 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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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 ...

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 ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

基于乙二醇玻璃化冷冻小鼠胚胎的超低温保存

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

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Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...