我们感谢 Cellerino, 泰隆 Genade, 安妮深色, 夏普, 米奇鲍威尔, 西蒙妮 Keil, Yumi 金, 帕特里克史密斯, 凯 Mathar 和 Valenzano 实验室的所有成员在最大的普朗克生物研究所为促进不同方面目前鳉鱼畜牧业的协议。

鱼在28°c 被上升在水循环系统 (参见水参量), 与 10-20% 每日水处置。三不同的坦克大小推荐: 0.8 升, 2.8 升和9.5 升。每罐接收恒定的水流2毫升/秒。
1. 试剂制备 (不含材料)
注: 非洲绿松石鳉鱼 (Nothobranchiusfurzeri) 可从已建立的实验室库存中提供。每年鳉鱼干燥的胚胎可以通过邮寄方式运送。在 8-30 °c 温度范围内运送胚胎是至关重要的。
<ol><li>在系统水中溶解1克/升腐植酸, 制备腐植酸 (孵化) 溶液。蒸压釜和贮存在4摄氏度, 长达10周。</li>
	<li>为制备 HUFA 卤虫浓缩, 每天在盐水虾溶液浓度为500µL/升的盐水虾孵化中加入 HUFA 富集。</li>
	<li>在以前的蒸压系统水中溶解100µL/升的亚甲基蓝溶液, 制备亚甲基蓝液。由于亚甲基蓝是光敏的, 保持溶液在黑暗的瓶子, 或覆盖与箔。存储在 RT。</li>
	<li>准备椰子纤维作为胚胎孵化的固体基质。或者, 使用过滤纸 (见1.5 节)。<ol>
<li>Presoak 椰子纤维用蒸馏水。蒸压釜和贮存在4摄氏度, 长达5周。</li>
		<li>在胚胎移植当天, 用湿润椰子纤维制备培养皿。</li>
		<li>填充一个90毫米直径的培养皿与椰子纤维下的油烟罩和旁边的火焰, 以减少污染的酵母和细菌。</li>
		<li>椰子纤维紧凑, 高度为1厘米, 无菌组织。把大部分的水分从椰子纤维上按在盘子上的纸巾上, 让纸张吸收多余的水。在火焰上加热一个金属勺子, 然后按下整个椰子纤维表面。这可以防止椰子纤维的真菌/细菌污染。</li>
	</ol>
</li>
	<li>准备过滤纸作为胚胎孵化的固体基质。<ol>
<li>在胚胎移植当天, 放置3层过滤纸盘, 适合90毫米培养皿。加入5毫升腐植酸溶液以保持湿度。</li>
	</ol>
</li>
</ol>2. 育种
<ol><li>
鳉鱼绿松石菌的选育 注意: 根据这项协议, 性成熟度达到4周后孵化和生育高峰之间 7-9 周。重要的是要注意, 生育力取决于喂养频率和食物质量;因此, 建议至少每天喂养两个饲养池, 以提高胚胎产量 (见5.6 节)。<ol>
<li>设置一个9.5 升繁殖罐。填充系统水并添加一条雄性和两只雌鱼。</li>
		<li>由于雄性非洲绿松石鳉鱼在交配期间表现出优势, 这可能会导致对雌性的骚扰, 所以选择一个比雌性体型稍小一点的雄性来减少交配压力, 增加生殖产量。成立了5周大的男性, 6/7 周大的女性。</li>
		<li>填充一个塑料容器 (10 x 10 x 5 厘米), 蒸压砂达到最终深度为 2-3 厘米, 并将砂箱放在繁殖槽的中心。</li>
		<li>让绿松石鳉鱼连续繁殖, 每周收获一次胚胎孵化。 注: 使用砂基板对集中式过滤系统构成挑战, 今后应采用替代方法替代。可能的替代方案可能是使用斑马鱼养殖坦克。</li>
	</ol>
</li>
	<li>
转基因育种 注: 用于注射的胚胎需要在单细胞阶段同步, 这需要在受精后立即收集。<ol>
<li>为了获得用于注射和生成转基因品系的一细胞级胚胎, 建立了一只雄性和两条雌鱼的繁殖槽 (与2.1 相同)。</li>
		<li>在胚胎采集前两天, 将雄性单独放在一个水箱中, 并保持雄性与成年雌性的视觉接触。</li>
		<li>在收集日, 将雄性和一个沙盒添加到繁殖罐中, 让它们产卵2小时。</li>
	</ol>
</li>
</ol>3. 胚胎饲养
<ol><li>
胚胎采集 注: 胚胎采集是通过从砂盒中筛选和收获胚胎进行的。在正常情况下, 每个砂箱应包含从30到200胚胎。<ol>
<li>在收集日, 从饲养箱中取出砂盒。将砂箱清空至过滤器 (0.9 毫米应变大小), 并用系统水冲洗。这可以通过一个大的坦克来收集沙子为热处理。</li>
		<li>将过滤器部分浸入系统水中, 轻轻旋转, 让胚胎聚集在中心。</li>
		<li>用10毫升的巴斯德吸管收集胚胎。</li>
		<li>将胚胎移植到90毫米培养皿中, 在40毫升的系统水中。</li>
		<li>在光显微镜下检查培养皿中的胚胎, 除去那些呈现破裂的绒毛膜和损伤的迹象。</li>
		<li>直接进行胚胎漂白。 注意: 每次鱼菌株都要使用一个过滤器, 以防止潜在的交叉应变胚胎污染。</li>
	</ol>
</li>
	<li>
胚漂白 注意: 胚胎漂白可防止鱼缸中的微生物污染孵化介质。<ol>
<li>漂白前, 使用一次性的巴斯德吸管从含有采集胚胎的培养皿中取出系统水。</li>
		<li>为防止不必要的真菌和细菌生长, 将50毫升新制备的 H2O2 (在蒸压系统水中1% 伏/v) 添加到所收集的胚胎中。</li>
		<li>在50毫升溶液中, 在90毫米培养皿中以低速速度摇动胚胎5分钟。</li>
		<li>将 H2O2解决方案与一次性的巴斯德吸管和洗涤胚胎三次为5分钟与50毫升亚甲基蓝溶液。移除亚甲基蓝溶液。</li>
		<li>在蒸压系统水中添加50毫升 H2O2 (1% 伏/v) 到胚胎并摇动5分钟。</li>
		<li>删除 H2O2解决方案, 并用50毫升亚甲基蓝溶液清洗三次5分钟。</li>
		<li>孵化28°c 的胚胎, 以增加同步胚胎发育, 最高密度为100胚每90毫米培养皿在40毫升亚甲基蓝溶液。 注意: 不要在漂白溶液中延长胚孵化。这可能会对鸡蛋绒毛膜造成损害, 并增加胚胎死亡率。胚胎漂白可能会导致鸡蛋绒毛膜的物理化学变化, 可能导致改变绒毛膜生理和孵化成功。</li>
	</ol>
</li>
	<li>
亚甲蓝胚孵化 注: 在亚甲蓝溶液中的液体孵化可以防止寄生虫生长, 并能检测死胚和未受精的卵。<ol>
<li>检查孵化的胚胎, 从培养皿中除去任何死胚 (由亚甲蓝染蓝), 以防止真菌和细菌污染影响活健康胚胎的生存。</li>
		<li>除去旧的亚甲基蓝溶液, 用新鲜溶液代替。</li>
		<li>将 Petri 盘返回到28°c 孵化器 (图 1A)。在 7-10 天内, 确保已发育的胚胎显示可见的黑色眼睛。将这些胚胎转移到椰子纤维或过滤纸固体基质介质 (图 1B)。</li>
		<li>保留未发育的亚甲蓝胚胎, 每日监测, 一旦黑眼睛发育, 就转移到固体基质培养基中。</li>
		<li>重复步骤 3.3. 1-3. 3.3 每天直到胚胎有肉眼可见的黑眼睛。 注意: 胚胎对亚甲基蓝的持续暴露可能会导致成年鱼类生理的长期变化。</li>
	</ol>
</li>
	<li>
胚胎移植到过滤纸上 注: 绿松石鳉鱼胚可在干燥基质上发育, 综述自然条件。此外, 干胚孵化使研究人员能够同步胚胎和孵化他们在同一天。<ol>
<li>随着发育的胚胎在 7-10 天内会有肉眼可见的黑眼睛, 使用一次性的巴斯德吸管或细弯镊子将胚胎从亚甲基蓝溶液转移到先前制备的滤纸板上。</li>
		<li>将胚胎与镊子分开5毫米, 每90毫米板可达100个胚胎 (图 1B)。</li>
		<li>用 parafilm 封住培养皿。</li>
		<li>孵化胚胎在28°c 2-3 周, 直到他们已经完全开发了金色的虹膜, 并准备孵化 (图 1C)。 注意: 不要延长孵化胚胎的时间超过2周, 因为它们的生存能力将大大减少。</li>
	</ol>
</li>
	<li>
椰子纤维的胚胎移植 注: 蒸压、无菌椰子纤维 (或有机泥炭苔藓) 可用作固体基质孵化的有效替代培养基。<ol>
<li>使用一次性的巴斯德吸管或细弯曲镊子将胚胎从亚甲基蓝溶液转移到现成的椰子纤维板上。</li>
		<li>将胚胎分布在5毫米之外, 每90毫米板最多100个胚胎 (图 1B)。</li>
		<li>用 parafilm 封住培养皿。</li>
		<li>孵化胚胎在28°c 2-3 周, 直到他们已经完全开发了金色虹膜 (例如在图 1C)。 注: 对于长期贮存 (最多一年), 在3天后从亚甲基蓝溶液到17摄氏度的固基板上转移胚胎。孵化胚胎直到它们发育成黑色的眼睛。</li>
	</ol>
</li>
</ol>4. 孵化绿松石鳉鱼
注: 绿松石鳉鱼胚胎可以成功孵化在腐植酸溶液14中.
<ol><li>使用细弯曲镊子, 仔细转移 50-100 开发的胚胎进入孵化盒填充的腐植酸溶液在4摄氏度。腐植酸溶液由系统水中的1克/升腐植酸组成。蒸压釜和贮存在4摄氏度, 长达10周。要确保所有的胚胎都完全浸入。腐植酸溶液必须浅, 不超过2厘米。 注: 腐植酸溶液的低温改善孵化和完全浸泡的胚胎在解决方案允许同步孵化。</li>
	<li>将孵化箱放入28°c 孵化孵化器。用盖子盖住孵化箱。要提供足够的曝气, 用油管与供气连接孵化箱。 注: 在孵化过程中没有足够的曝气导致高率的鱼苗无法填满气体膀胱 ("肚皮滑块" 表型, 见5.1 节中的说明)</li>
	<li>从孵化后的次日起, 在孵化箱中保持足够的水质, 每天加蒸压水系统的比例为 1:1, 保持最后深度2厘米。</li>
	<li>将孵化胚胎转移回固体基质, 并在一周后尝试孵化。 注: 孵化后, 绿松石鳉鱼很容易吸收和食用活食物。为最佳生长, 饲料鱼苗每天两次与过剩新鲜孵化盐水虾 (卤咸藻)。充分饱足的标志是橙色的腹部后, 每喂养 10-15 分钟。每天用巴斯德吸管抽出多余的、吃剩的和分解过的盐水虾。</li>
</ol>5. 饲养幼鱼和成年鱼类
<ol><li>孵化后五天, 将幼仔移至水循环系统。使用一次性塑料吸管 (或塑料勺), 小心地转移每 0.8 L 坦克配备400µm 油炸屏幕 (图 2) 的五青少年。 注: 有可能部分少年鳉鱼不会填满气膀胱, 导致典型的 "腹部滑块" 表型, 其特征是鱼没有达到适当的浮力, 迫使他们连续游泳, 造成严重畸形成年鱼。这些鱼不能用于生存化验或有效育种, 需要被审查。</li>
	<li>每日两次喂养幼鱼, 新鲜孵化的盐水虾超过14天后孵化。每天从每罐底部抽出碎片。</li>
	<li>在14天的年龄, 转移幼鱼到2.8 升坦克配备了850µm 油炸屏幕。从这一点开始, 标签每个坦克与鱼的 ID, 表明舱口日期, 应变信息, 鱼类性别和鱼类识别号码 (图 3)。为生存化验, 单独地房子每条鱼在单坦克从这一点开始。</li>
	<li>在接下来的7天里, 每条鱼用2毫升的盐水虾每天喂两次幼仔。在这个阶段鱼可以补充与 1-3 活的血液蠕虫 (如果血液蠕虫幼虫是太大为鱼, 砍他们入更小的片断用剃刀刀片)。为防止水质恶化, 每周抽出两次吃剩的食物和额外的废物。</li>
	<li>从孵化3周后, 从罐体背面取出油炸筛, 每天两次喂鱼2毫升盐水虾和0.5 毫升的血蠕虫。在这个阶段, 青少年应该达到1厘米的身体大小, 应该能够摄取全尺寸的血蠕虫。</li>
	<li>在4周的年龄, 每条鱼每天两次喂养2毫升的盐水虾和1毫升的血蠕虫。雌性可以被共同安置在密度高达3女性每 2.8 L 坦克。</li>
	<li>在这个阶段确保鱼达到完全性成熟。检查是否存在大背, 肛门和尾鳍的迹象, 男性和圆形腹部满卵的雌性。 注: 在单个贮罐中饲养成年鱼以进行生存队列研究可能对鱼类的行为和健康产生负面影响。然而, 群体住房的生存队列研究增加了重要的混杂因素, 由于建立社会优势和男性领土, 导致严格的社会等级制度。</li>
</ol>6. 喂养
注: 实验室绿松石鳉鱼可喂养婴儿盐水虾 (卤虫藻幼体) 和血蠕虫 (摇幼虫) 的组合.绿松石鳉鱼鱼苗只喂养婴儿盐水虾。幼鱼和成年鱼类每天喂两次盐水虾和血蠕虫 (图 2)。理想情况下, 鱼可以每天喂食多次, 超过本议定书所示的2喂养。
<ol><li>
盐水虾养殖
	<ol>
<li>将10升反渗透 (RO) 水和350克红海盐添加到盐水虾孵化中, 用曝气管曝气溶解。</li>
		<li>用5毫升高不饱和脂肪酸 (HUFA) 来丰富培养。</li>
		<li>在孵化液中加入20克盐水虾囊肿。检查盐水虾囊肿不漂浮在水面上, 并确保适当的氧合和循环的文化。 注: 每日整除数的干盐水虾囊肿可储存在4摄氏度。</li>
		<li>第二天下午, 供应的文化与另一整除5毫升的 HUFA。</li>
	</ol>
</li>
	<li>
收获孵化的盐水虾 注: 从起动培养到36小时后, 盐水虾就可以收割 (二龄期)。<ol>
<li>收集 5 L 的文化在一个容器中使用水龙头底部的孵化器, 让坐10分钟。</li>
		<li>10分钟后, 从5升容器的顶部除去盐水虾壳 (棕色), 并通过网格过滤孵化的盐水虾 (橙色)。注意排除在容器底部发现的沉淀物, 因为它含有非孵化蛋和死盐水虾。</li>
		<li>用 RO 水将孵化后的盐水虾冲洗成2升的容器, 让它坐10分钟。</li>
		<li>10分钟后, 再用滤网过滤盐水虾, 并在2升的 RO 水中采集。</li>
		<li>重复前三步骤, 直到盐水虾解决方案没有孵化囊肿和盐水虾壳。</li>
		<li>将盐水虾从2升容器中转移到挤瓶中进行喂养。 注: 养殖盐水虾是相当健壮和可靠的。但是, 为了避免盐水虾在孵化失败的情况下短缺, 可以使用较小的 (500 毫升) 备份 hatchers。</li>
	</ol>
</li>
	<li>
设置备份孵化器
	<ol>
<li>用曝气法将17.5 克的红海盐溶解在500毫升的 RO 水中。</li>
		<li>以500µL 的 HUFA 丰富文化。</li>
		<li>加入2克盐水虾囊肿。</li>
		<li>在 18-24 h 以后, 再供应文化以500µL HUFA。 注: 盐水虾准备在24小时后收割。</li>
	</ol>
</li>
	<li>
活血蠕虫的制备
	<ol>
<li>在喂食之前, 用 RO 水过滤适量的血虫。 注: 活血蠕虫可以储存在4°c 7-10 天。</li>
		<li>用少量的 RO 水将血蠕虫冲洗成塑料容器。</li>
		<li>用一个塑料巴斯德吸管 (狭窄的尖端去除), 拿起血液蠕虫混合物喂养。 注: 用来自联合国控制源的活食物喂养实验室鳉鱼菌落增加了来自寄生虫和潜在致病微生物群落的外部污染物的风险。今后应开发一种特殊的不育鱼饲料。</li>
	</ol>
</li>
</ol>7. 鳉鱼实验室菌株基因分型
注意: 为了区分绿松石鳉鱼菌株, 以及确定每个菌株中的性别, 可以使用特定的基因 (微卫星) 标记 9 ( 表1)。
<ol><li>
采样
	<ol>
<li>把鱼牢牢地放在湿海绵顶部的网中。</li>
		<li>拭子 2-3 鳞片从鱼体从笼盖到尾翅使用棉签。</li>
		<li>在含氢氧化钠溶液的1毫升管 (200 µL 0.5 摩尔/L 氢氧化钠, 1% β巯基乙醇, 0.5% 聚乙烯醇吡咯烷酮) 中, 选取从拭子中提取的鳞片并转移 2-3 鳞片。</li>
		<li>在十五年代旋转 PCR 管以确保鳞片完全浸入氢氧化钠溶液中。</li>
	</ol>
</li>
	<li>
基因组 DNA 分离
	<ol>
<li>在95摄氏度孵化样品20分钟。</li>
		<li>冷却在 RT, 中和样品以1/10 容量 1 M 三 HCl, pH 8.0。</li>
		<li>以全速离心样品5分钟。</li>
	</ol>
</li>
</ol>8. 水参数
注: 在目标物种的整个生命周期内, 其预期用途为成人分型的生物体的畜牧业需要高度稳定的畜牧业条件。因此, 培养水生生物, 如绿松石鳉鱼, 必须严格控制水的参数。水循环, 再加四步水过滤, 确保了可靠的基础, 以达到对水参数的控制, 提供所有的水箱, 随着时间的推移相同的水条件。建议将系统水从反渗透 (RO) 水中重新使用, 并加入商业海水盐和碳酸氢钠。
<ol><li>水循环系统方案: 第一, 从鱼缸中流出的废水通过固体微粒金属过滤器来捕获所有未被吃掉的食物碎片和更大的微粒。金属过滤器每周冲洗三次;其次, 在第一次机械过滤后, 水被输送到大污水坑中, 然后注入到生物滤池中, 细菌将氨转化为亚硝酸盐和硝酸盐;第三, 从生物滤池中, 水被泵送到 25-µm 过滤套管, 它可以捕获更细的颗粒。最后, 水流经紫外线 (紫外线) 灯, 从细菌和病毒中消毒水。经过这四步, 过滤后的水返回到鱼缸。</li>
	<li>为了减少微生物在水箱中的生长, 防止硝酸盐积聚, 降低总盐度, 每天处理 10-20% 的系统水。</li>
	<li>保持水温恒定在28摄氏度, 水 pH 常数在7.0 到7.5 范围内。</li>
	<li>虽然鳉鱼容忍广泛的盐度, 以避免 oodinosis, 保持电导率范围内 650-710 微西门子。一年长达12小时的光/暗循环确保了群体的健康和生产力。 注: 鳉鱼可耐受1500微西门子的水电导率。</li>
</ol>

<ol>
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R., Aboobaker, A., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+aging+model+systems+and+why+we+need+them.">Non-canonical aging model systems and why we need them.</a> EMBO J. 36 (8), 959-963 (2017).</li><li>Harel, I., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Turquoise+Killifish:+A+Model+for+Exploring+Vertebrate+Aging+and+Diseases+in+the+Fast+Lane.">The African Turquoise Killifish: A Model for Exploring Vertebrate Aging and Diseases in the Fast Lane.</a> Cold Spring Harb Symp Quant Biol. 80, 275-279 (2015).</li><li>Smith, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+life+span+by+the+gut+microbiota+in+the+short-lived+African+turquoise+killifish.">Regulation of life span by the gut microbiota in the short-lived African turquoise killifish.</a> Elife. 6, (2017).</li><li>Cellerino, A., Valenzano, D. R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+bush+to+the+bench:+the+annual+Nothobranchius+fishes+as+a+new+model+system+in+biology.">From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology.</a> Biol Rev Camb Philos Soc. 91 (2), 511-533 (2016).</li><li>Kim, Y., Nam, H. G., Valenzano, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short-lived+African+turquoise+killifish:+an+emerging+experimental+model+for+ageing.">The short-lived African turquoise killifish: an emerging experimental model for ageing.</a> Dis Model Mech. 9 (2), 115-129 (2016).</li><li>Blazek, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+intraspecific+divergence+in+life+span+and+aging+of+African+annual+fishes+along+an+aridity+gradient.">Repeated intraspecific divergence in life span and aging of African annual fishes along an aridity gradient.</a> Evolution. 71 (2), 386-402 (2017).</li><li>Terzibasi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+differences+in+aging+phenotype+between+strains+of+the+short-lived+annual+fish+Nothobranchius+furzeri.">Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri.</a> PLoS One. 3 (12), e3866 (2008).</li><li>Kirschner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+quantitative+trait+loci+controlling+lifespan+in+the+short-lived+fish+Nothobranchius+furzeri--a+new+vertebrate+model+for+age+research.">Mapping of quantitative trait loci controlling lifespan in the short-lived fish Nothobranchius furzeri--a new vertebrate model for age research.</a> Aging Cell. 11 (2), 252-261 (2012).</li><li>Valenzano, D. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Turquoise+Killifish+Genome+Provides+Insights+into+Evolution+and+Genetic+Architecture+of+Lifespan.">The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.</a> Cell. 163 (6), 1539-1554 (2015).</li><li>Harel, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platform+for+rapid+exploration+of+aging+and+diseases+in+a+naturally+short-lived+vertebrate.">A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.</a> Cell. 160 (5), 1013-1026 (2015).</li><li>Valenzano, D. R., Sharp, S., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-Mediated+Transgenesis+in+the+Short-Lived+African+Killifish+Nothobranchius+furzeri,+a+Vertebrate+Model+for+Aging.">Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging.</a> G3. 1 (7), 531-538 (2011).</li><li>Harel, I., Valenzano, D. R., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+engineering+approaches+for+the+short-lived+African+turquoise+killifish.">Efficient genome engineering approaches for the short-lived African turquoise killifish.</a> Nat Protoc. 11 (10), 2010-2028 (2016).</li><li>Polacik, M., Blazek, R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+breeding+of+the+short-lived+annual+killifish+Nothobranchius+furzeri.">Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri.</a> Nat Protoc. 11 (8), 1396-1413 (2016).</li><li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li></ol>

所有的作者都宣称没有竞争的金融和非金融利益。

我们描述了一项关于绿松石鳉鱼实验室培养的协议, 包括胚胎采集, 孵化, 以及成年鱼类的住房, 饲养和饲养。我们的议定书专门针对从事成年鱼类研究的实验室, 特别是关于衰老和寿命的实验研究。绿松石鳉鱼可以在标准的斑马鱼设施上饲养;然而, 鳉鱼畜牧业的重要方面不同于标准的斑马鱼护理 16.这些调整包括从盐水虾只饮食的早期过渡到饮食补充富含蛋白质的血液蠕虫, 以及胚胎孵化的具体步骤, 包括液体和干燥孵化阶段。
协议中的关键步骤包括在 8-30 °c 温度范围内运送胚胎。在繁殖的情况下, 生育力取决于摄食频率和食物质量;因此, 我们建议至少每天喂养两个饲养池, 以提高胚胎产量 (见5.6 节)。在胚胎漂白过程中, 不要在漂白溶液中延长胚孵化。这可能会对鸡蛋绒毛膜造成损害, 增加胚胎死亡率。当用亚甲蓝孵化胚胎时, 不要延长准备孵化胚胎的潜伏期超过2周, 因为它们的生存能力将大大减少。为孵化绿松石鳉鱼, 腐植酸溶液的低温改善孵化和完全浸泡的胚胎在解决方案允许同步孵化。没有足够的曝气在孵化期间导致高率的鱼苗无法填满气体膀胱 ("肚皮滑块" 表型, 见5.1 节附注)。
育种议定书的限制包括使用对集中过滤系统构成挑战的砂基板, 今后应改用替代方法。可能的替代方案可能是使用斑马鱼养殖坦克。胚胎漂白可能会导致鸡蛋绒毛膜的物理化学变化, 可能导致改变绒毛膜生理和孵化成功。胚胎持续暴露于亚甲基蓝可能会引起成年鱼类生理的长期变化。在单独的坦克中饲养成年鱼进行生存队列研究可能会对鱼类的行为和健康产生负面影响。然而, 群体住房的生存队列研究增加了重要的混杂因素, 由于建立社会优势和男性领土, 导致严格的社会等级制度。因此, 我们认为, 分离雄性鱼类进行生存研究是一个合理的妥协。喂养实验室鳉鱼菌落和来自非控制源的活食物增加了来自寄生虫和潜在致病微生物群落的外部污染物的风险。今后应开发一种特殊的不育鱼饲料。
今后对该议定书的改进将集中在受控的非生活饮食, 这仍然导致 3-4 周内完成性成熟。总之, 我们的协议为绿色鳉鱼实验室培养到广泛的科学界提供了方便。

由于短寿命和快速生命周期, 绿松石鳉鱼正在迅速成长为一个有前途的新的模式生物在生物学 1, 2, 3.这个物种的特点是一个独特的生命周期的硬骨鱼类, 包括胚胎滞育, 快速性成熟, 和延长后生殖生命阶段4, 5.最近的工作有助于阐明这一物种的生物学在圈养和野生6,7。绿松石鳉鱼生活在津巴布韦和莫桑比克的非洲大草原雨季期间形成的季节性淡水体中。在旱季, 由于不缺水, 胚胎在干燥的泥浆中存活, 这是一种叫做滞育的抗应力生命期。
该物种的遗传图谱已生成8,9, 最近他们的基因组已经测序和组装10,11。一些近交的实验室鱼类菌株已经开发, 转基因和基因组编辑通过 CRISPR/Cas9 已成为在这个物种,事实上促进绿松石鳉鱼作为一个竞争性实验室脊椎动物模型生物12,13,14。
虽然已经为这个物种发布了一个实验室协议15, 但在本议定书中, 我们制定了一份全面的实验实验室指南, 专门针对调查衰老和生存的研究。本议定书使已经熟悉斑马鱼和鳉畜牧业的研究人员通过最小数量的关键调整来精通绿松石鳉鱼畜牧业。同时, 该议定书还为研究人员提供了在渔业方面没有经验的重要工具, 以提高蓬勃发展的绿松石鳉鱼殖民地。

<table>
	<tbody>
		<tr>
			<td>Probe calibration buffer solution pH=7.0</td>
			<td>Roth</td>
			<td>A518.1</td>
			<td>1L buffer solution pH=7.0 to calibrate water system pH-electrode</td>
		</tr>
		<tr>
			<td>Probe calibration buffer solution pH=4.0</td>
			<td>Roth</td>
			<td>P712.1</td>
			<td>1L buffer solution pH=4.0 to calibrate water system pH-electrode</td>
		</tr>
		<tr>
			<td>Conductivity standard</td>
			<td>VWR</td>
			<td>83607.260</td>
			<td>500 mL Conductivity standard 1,413 uS/cm to calibrate water system conductivity-electrode</td>
		</tr>
		<tr>
			<td>Easy Strips Test 6in1</td>
			<td>JBL</td>
			<td>2533900</td>
			<td>Test strips for determination chlorine values of system water</td>
		</tr>
		<tr>
			<td>Ammonia Test</td>
			<td>JBL</td>
			<td>2536500</td>
			<td>Test to determine ammonia content of system water</td>
		</tr>
		<tr>
			<td>Red Sea Salt</td>
			<td>Red Sea</td>
			<td></td>
			<td>22 kg bucket</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>VWR</td>
			<td>27780.360</td>
			<td></td>
		</tr>
		<tr>
			<td>Humic acid</td>
			<td>Sigma- Aldrich</td>
			<td>53680-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>New HUFA Artemia enrichment</td>
			<td>ZM Systems UK</td>
			<td></td>
			<td>75g bottle</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Roth</td>
			<td>AE64.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma- Aldrich</td>
			<td>31642-1L</td>
			<td>30% (w/w)</td>
		</tr>
		<tr>
			<td>Cononut fiber</td>
			<td>Dragon</td>
			<td>ZCS010</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE healthcare</td>
			<td>3030-690</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol pure</td>
			<td>VWR</td>
			<td>20821.467</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Silica sand</td>
			<td>local pet shop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artemia Eggs Premium Grade</td>
			<td>Sanders</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bloodworm</td>
			<td>local distributor</td>
			<td></td>
			<td>Poseidon Aquakultur Germany</td>
		</tr>
		<tr>
			<td>dNTPs solution mix</td>
			<td>Biolabs</td>
			<td>N04472</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>Invitrogen</td>
			<td>18038-042</td>
			<td>5U/uL</td>
		</tr>
		<tr>
			<td>PCR 10x Buffer</td>
			<td>Invitrogen</td>
			<td>18038-042</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Invitrogen</td>
			<td>18038-042</td>
			<td>50mM</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma- Aldrich</td>
			<td>S8045-500g</td>
			<td>50mM</td>
		</tr>
		<tr>
			<td>Tris-HCl, pH=8.0 solution</td>
			<td>Sigma- Aldrich</td>
			<td>T2694-1L</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>HCl 37%</td>
			<td>Sigma- Aldrich</td>
			<td>H1758-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquatic housing system</td>
			<td>Aquaneering</td>
			<td></td>
			<td>Central filtration equipped aquatic system</td>
		</tr>
		<tr>
			<td>Fish tanks</td>
			<td>Aquaneering</td>
			<td></td>
			<td>volume: 0.8L, 2.8L, 9.5L; equipped with baffles, fry mesh and lids</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-100</td>
			<td>model 5000</td>
		</tr>
		<tr>
			<td>Microbiological incubator</td>
			<td>Thermo Scientific, Heratherm</td>
			<td>50125882</td>
			<td>model IMC18; for storage embryos in the liquid phase, set to t=27-28&#176;C</td>
		</tr>
		<tr>
			<td>Cooling Incubator</td>
			<td>Binder</td>
			<td>9020-0209</td>
			<td>model KT115; for storage embryos in the solid phase, set to t=27-28&#176;C</td>
		</tr>
		<tr>
			<td>Hatching incubator</td>
			<td>Thermo Scientific, Heratherm</td>
			<td>51028114</td>
			<td>model OGS180; for embryos hatching, set to t=27-28&#176;C</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td></td>
			<td>model M80</td>
		</tr>
		<tr>
			<td>Breeding sand/hatching boxes</td>
			<td>Roth</td>
			<td>1598.1</td>
			<td>1000mL</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473</td>
			<td>92x16mm</td>
		</tr>
		<tr>
			<td>50mL Conical tube</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical tube</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Plastic Pasteur pipette</td>
			<td>Roth</td>
			<td>EA71.1</td>
			<td>2mL; For fish feeding with bloodworms, or embryos selection cut off the tip to open 3-4mm diameter</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Sarstedt</td>
			<td>86.1689.001</td>
			<td>50mL</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Henke Sass Wolf</td>
			<td>4100-000V0</td>
			<td>10mL</td>
		</tr>
		<tr>
			<td>Metal strainer</td>
			<td></td>
			<td></td>
			<td>fineness &#60;1mm; for embryos collection</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0508-5/45-PO</td>
			<td>type5/45; for embryos transfer</td>
		</tr>
		<tr>
			<td>25L Brine shrimps hatcher</td>
			<td>Aquaneering</td>
			<td>ZHBS25</td>
			<td>main hatcher</td>
		</tr>
		<tr>
			<td>500mL Brine shrimps hatcher</td>
			<td>JBL</td>
			<td>6106100</td>
			<td>model Artemio 1; backuo hatcher</td>
		</tr>
		<tr>
			<td>Narrow-mesh fish nets</td>
			<td>JBL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sand beaker</td>
			<td>VWR</td>
			<td>BURK7102-5000</td>
			<td>5000mL</td>
		</tr>
		<tr>
			<td>Brine shrimps separation beaker</td>
			<td>VWR</td>
			<td>BURK7102-2000</td>
			<td>2000mL</td>
		</tr>
		<tr>
			<td>Plastic zipper bag</td>
			<td>Roth</td>
			<td>P279.2</td>
			<td>for dead fish storage</td>
		</tr>
		<tr>
			<td>Pipetboy</td>
			<td>Integra</td>
			<td>155000</td>
			<td>model Pipetboy acu2</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>P-Lab</td>
			<td>P701605</td>
			<td></td>
		</tr>
		<tr>
			<td>Air tubing</td>
			<td>www.zajac.de</td>
			<td>AQ380</td>
			<td>4-6 mm diameter</td>
		</tr>
		<tr>
			<td>1L Glass bottle</td>
			<td>VWR</td>
			<td>215-1595</td>
			<td></td>
		</tr>
		<tr>
			<td>2L Glass bottle</td>
			<td>VWR</td>
			<td>215-1596</td>
			<td></td>
		</tr>
		<tr>
			<td>500mL Squeeze bottle</td>
			<td>Roth</td>
			<td>K665.1</td>
			<td>for fish feeding with brine shrimps</td>
		</tr>
		<tr>
			<td>120 micron brine shrimps strainer</td>
			<td>Florida Aqua Farms</td>
			<td>BB-PC2</td>
			<td>for brine shrimps/bloodworm collection</td>
		</tr>
		<tr>
			<td>Finish filter socks</td>
			<td>Aquaneering</td>
			<td>MFVB025C</td>
			<td>25 micron</td>
		</tr>
		<tr>
			<td>Central filtration fish housing system</td>
			<td></td>
			<td></td>
			<td>Aquaneering, Techniplast, Aquatic Habitats, Aqua Schwarz</td>
		</tr>
	</tbody>
</table>

a protocol for laboratory housing of turquoise killifish (nothobranchius furzeri)

开发用于实验目的的非模型实验室鱼类的畜牧业做法, 大大得益于建立参考鱼类模型系统, 如斑马鱼和鳉。近年来, 一条新兴的鱼类--绿松石鳉鱼 (Nothobranchiusfurzeri)--已被越来越多的生物老化和生态学领域的研究小组采用。由于 4-8 月的圈养寿命, 这一物种是在圈养中饲养的最短的脊椎动物, 允许科学界在短时间内对实验性干预进行试验, 从而导致老化率和预期寿命的变化。鉴于这一物种独特的生物学特性, 其特点是胚胎滞育、爆炸性性成熟、明显的形态学和行为性异形以及它们相对较短的成年寿命--临时畜牧业的做法迫在眉睫. 需求。该议定书报告了一系列关键的畜牧业措施, 允许最佳的绿松石鳉鱼实验室护理, 使科学界能够采用这个物种作为一个强大的实验动物模型。

绿松石鳉鱼适当的畜牧业导致中位存活率在 GRZ 菌株的 12-18 周之间 (如图4A)。中位存活率的变化取决于饮食、喂养频率和住房温度条件。恶劣的畜牧业导致了生存曲线的出现, 从而增加了早期死亡率和重复的, 突然下降的生存时间, 特点是几个拐点 (图 4B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57073/57073fig1.jpg"> 图 1: 具有各自孵化基质的代表性胚胎发育阶段。(A)新鲜收集的胚胎, 孵化在28摄氏度的孵化器中的亚甲基蓝溶液中。(B)胚胎准备转移到固体介质中, 无论是过滤纸还是椰子纤维。(C)胚胎准备孵化, 显示典型的金色虹膜。刻度条等于1毫米.<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57073/57073fig2.jpg"> 图 2: 绿松石鳉鱼孵化后发育阶段。<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57073/57073fig3.jpg"> 图 3: 从阶段3开始的鱼的代表性鱼 ID 标记.<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57073/57073fig4.jpg"> 图 4:70 雄性绿松石鳉鱼的代表性生存曲线。(A)实验室饲养的绿松石鳉鱼的典型生存曲线。(B)在最佳畜牧业条件 (黑色) 和不良畜牧业 (红色和蓝色) 下养殖的鱼类生存曲线的比较。虚线红色水平线表示50% 生存, 相交生存曲线在中值寿命 (在 x 轴上表示)。<a href="https://cloudflare.jove.com/files/ftp_upload/57073/57073fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Id</td>
			<td colspan="2">前进底漆</td>
			<td colspan="2">反向底漆</td>
			<td>尺寸范围 (bp)</td>
		</tr>
<tr>
<td>* NfuSU0007</td>
			<td colspan="2">GGCTAAGCCTTGCTGACAGA</td>
			<td colspan="2">CAGGGAGCTGAAAACCTCAG</td>
			<td>166-214</td>
		</tr>
<tr>
<td>* NfuSU0010</td>
			<td colspan="2">CGCAGTCTGATCAAATCGTGT</td>
			<td colspan="2">TGTTTGAAGGTTCACATTCATTATC</td>
			<td>220-272</td>
		</tr>
<tr>
<td>NfuSU0016</td>
			<td colspan="2">CATGGCTAAACCGTGATGAA</td>
			<td colspan="2">GAAGGACGCCAGCTATGAAG</td>
			<td>209-240</td>
		</tr>
<tr>
<td>NfuSU0022</td>
			<td colspan="2">AACACAGCTCTCGTAAGGAGGTA</td>
			<td colspan="2">TTCAGACTTGTCTTACTACCATGTTT</td>
			<td>198-238</td>
		</tr>
<tr>
<td>NfuSU0027</td>
			<td colspan="2">TCCAGCTGAATCGGTAATGA</td>
			<td colspan="2">AAACTCGAGGGTGCAATCTG</td>
			<td>164-226</td>
		</tr>
<tr>
<td>NfuSU0049</td>
			<td colspan="2">CTGGACAAAGTGCCAATCAC</td>
			<td colspan="2">CTCCCACAGTCCCAAAACAT</td>
			<td>196-197</td>
		</tr>
<tr>
<td>NfuSU0050</td>
			<td colspan="2">CCAGAATGAACAATACTCAGATCAA</td>
			<td colspan="2">GCAGCTTAGTTTAATGATATCACAATG</td>
			<td>252-295</td>
		</tr>
<tr>
<td>NfuSU0060</td>
			<td colspan="2">CTAGCCACTCCCCTGGTTTA</td>
			<td colspan="2">CCGTCACGATGTGCTGATAC</td>
			<td>216-248</td>
		</tr>
<tr>
<td>NfuFLI0030</td>
			<td colspan="2">CAGAAGCTAAAGGCCAGACG</td>
			<td colspan="2">GGGAAACAATAGGGAACCAC</td>
			<td>174-205</td>
		</tr>
<tr>
<td>* NfuFLI0091</td>
			<td colspan="2">ACGCTGACTCTACCCAGTC</td>
			<td colspan="2">CTGCCTGCTACTGACAATG</td>
			<td>355-373</td>
		</tr>
<tr>
<td colspan="6">* 性别决定标记</td>
		</tr>
</tbody></table>表 1: 菌株鉴定的基因型引物。

Watch this Scientific Journal Video about A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri at JoVE.com

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

在集中式水过滤系统中, 可以将绿松石鳉鱼的实验室房屋扩大到室内, 并有效地提高数以千计的个体鱼类, 同时采用与标准斑马鱼设施相同的基础设施。在这里, 我们详细列出了一个标准化的程序, 允许有效的鳉鱼维护。

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

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A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

16.7K 次观看.  Max Planck Institute for the Biology of Ageing. 在集中式水过滤系统中, 可以将绿松石鳉鱼的实验室房屋扩大到室内, 并有效地提高数以千计的个体鱼类, 同时采用与标准斑马鱼设施相同的基础设施。在这里, 我们详细列出了一个标准化的程序, 允许有效的鳉鱼维护。

视频: A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

培养大鼠颈上神经节（SCG）的交感神经元的议定书

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1四聚体的生产协议

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

基于计算机的蛋白质结构与功能预测议定书

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

为ArcturusXT仪器SIVQ-LCM协议

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...