我们感谢秀丽遗传学中心 (由国立卫生研究院 (NIH) 资助的研究基础设施项目 P40 OD010440) 为菌株。作者要感谢 Roelens 和 Eli Lessman, 为我们提供建设性的反馈, 并在 CCNY, 纽约市立大学的马诺河实验室使用他们的实验室的一部分拍摄和他们的帮助。这项工作得到了 PSC-纽约市立大学奖 TRADB-46-113 和 NIH 赠款1SC2GM118275-01 的支持。多发性硬化症得到了加拿大卫生研究院 (卫生研究院) 博士后奖学金的部分支持, 杨永强得到了 NIH/研究院增加补助金 GM062981 的支持。

1. 设置rec-8的 rna 干扰 (图 2中的1天 3)
此协议是从卡马斯和 Ahringer30中修改的。
<ol><li>为在大肠杆菌中诱导rec-8 dsRNA 表达式, 准备线虫生长培养基 (NGM) 琼脂板31补充最终浓度为1毫米 isopropyl-β-D-2-thiogalactopyranoside (IPTG) 和100µg/毫升西林.储存在黑暗中4°c, 直到使用, 长达4周。</li>
	<li>条纹 HT115 细菌携带rec-8 (W02A2.6) 克隆从 Ahringer 实验室库30 rec-8克隆到 Luria 肉汤 (LB) 板, 辅以100µg/毫升氨苄西林和50µg/毫升四环素。在37摄氏度的振动筛中一夜之间生长。</li>
	<li>在1天, 接种单一殖民地从新的有条纹的单一殖民地的 HT115大肠杆菌细菌携带rec-8 rna 克隆成4毫升 LB 含有100µg/毫升氨苄西林和50µg/毫升四环素。在37摄氏度的摇床或滚筒鼓中一夜之间生长细菌培养。</li>
	<li>次日早晨 (2 天), 在 HT115 细菌培养中, 通过增加1毫米 IPTG 的最后浓度, 并在37摄氏度上晃动40分钟, 诱导生产双绞链 (ds) RNA, 用于rec-8 W02A2.6。</li>
	<li>诱导后, 种子 NGM/IPTG 板与 100-200 µL HT115 rec-8细菌和贮存板在室温下夜间 (3 天)。</li>
	<li>次日早上 (4 天), 将所需的线虫菌株添加到诱导的 HT115 rec-8细菌板块, 如下面步骤2.1 所述。</li>
</ol>2. 生成和隔离 Tetraploids (图 2中的天 4-16)
<ol><li>在4天, 地方 3-4 幼 L4 (第四幼虫) 阶段雌雄同体的期望双菌株的 NMG/IPTG 板块种子与 HT115 rec-8细菌, 已诱导前一天 (步骤 1.5, 以上)。在黑暗中生长15摄氏度的线虫。</li>
	<li>重复步骤1.3 和1.4 开始3天后, 步骤 2.1, 这将是三天前的第一个后裔 (F1s) 从该步骤成为 L4s。这个步骤的时间将取决于线虫菌株在15摄氏度的生长速度。一些双倍变种菌株是缓慢的种植者, 这个时间将需要微调, 以隔离 tetraploids。</li>
	<li>在8天 (经过4天的rec-8 rna 喂养治疗), 转移 20 (2 雌雄同体/培养皿) 的 F1 (第一子代) L4 雌雄同体生长在 HT115 rec-8细菌到新鲜诱导 HT115 rec-8 rna 干扰和允许他们自我施肥。<ol>
<li>或者, 将在 HT115 rec-8细菌中生长的 F1 L4 雌雄同体的20转移到新诱导的 HT115 rec-8 , 并与同一菌株的未经处理的雄性一起 (2 雌雄同体 4-6 男性/板块)。</li>
	</ol>
</li>
	<li>在13天, 开始筛查 F2 (第二代子代) 的后代, 出现长 (隆) 或整体大于野生类型;然后将单个的离子动物转移到正常的 OP50 或 HB101 菌株的大肠杆菌细菌。继续筛查 F3 (第三子代) 后代;然而, F3 的后代从同一板块不能被认为是独立的菌株, 如果他们成为建立, 因为他们可能是兄弟姐妹从同一已经建立的 F2 母亲。 注意: 假定的 tetraploids 很容易识别, 因为它们明显长于 diploids。野生型雌雄同体比 tetraploids 短两三。由于它的时间较长, 二、四倍体杂交体在向前移动时会产生一个额外的转弯, 它通过从头部到尾部生成正弦体弯曲波(图 3A)。</li>
	<li>允许离子蠕虫自我受精和繁殖通过采摘离子后代, 直到只有在播种的后代是在没有rec-8的 rna 干扰治疗。这可能需要另外三代人。离子蠕虫通常是无菌的, 不会产生后代。</li>
</ol>3. 验证二、四倍体杂交菌株 (电影 1和图 3A, B)
注意: 二、四倍体杂交菌株可以通过计数未受精卵母细胞中的染色体数量来验证。荧光显微镜可用于筛查未受精双卵母细胞 (在减数分裂分裂之前) 染色体对的数量, 如果该菌株具有染色体的荧光标记。在缺乏荧光染色体标记的情况下, 可以通过固定线虫和用 DNA 染料染色来筛选二、四倍体杂交线虫;见下文为乙醇固定和全动物 4′-, 6-Diamidine--2′-苯基吲哚二盐酸盐 (DAPI) 染色的协议。
<ol><li>全动物 DAPI 染色 注: 携带12条连接染色体对的二、四倍体杂交菌株可以通过 DAPI 染色这些动物并计算其未受精卵母细胞的 DAPI 体数量来验证。<ol>
<li>将5到10µL M9 缓冲区31放在显微镜幻灯片上, 然后将 6-10 线虫转移到下拉。</li>
		<li>在解剖显微镜下, 从水滴中提取大部分 M9, 而不使用无起毛的清洁组织去除线虫。然后, 立即添加10µL 下降90% 乙醇到蠕虫。允许蠕虫完全干燥, 但不超过几秒钟。</li>
		<li>一旦乙醇蒸发 (这可以看到, 因为它发生在解剖显微镜下), 增加10µL 90% 乙醇的蠕虫。</li>
		<li>重复步骤3.1.3 三次。</li>
		<li>一旦最后一滴乙醇蒸发, 添加6µL 的 DAPI 或赫斯特染料的建议最终集中在安装介质的选择 (例如, 1:1, 000 稀释 2 ng/µL DAPI 库存浓度 M9)。对于长期储存的幻灯片, 0.5% 对氨基苯二胺溶解在20毫米三盐酸, pH 8.8, 在90% 甘油作为一个 antifade 溶液, 而不是仅 M9, 可使用。</li>
		<li>用盖玻片盖上滑梯上的蠕虫, 用指甲油封住盖玻片的边缘。分数在荧光显微镜 (步 3.1.7) 至少10分钟后添加盖玻片。没有 antifade 的幻灯片可以在评分前的4摄氏度储存几天, 但是荧光的质量在几天后开始下降。</li>
		<li>使用荧光显微镜在100X 放大, 评分的单一 DAPI 体 (想必是单染色体对) 在最成熟的未受精卵母细胞, 这是直接相邻的蝗受精囊, 并尚未进入蝗受精囊或子宫。<ol>
<li>为了在卵母细胞核内单独 DAPI 体, 使用显微镜的精细聚焦在计数期间从卵母细胞细胞核的顶端缓慢移动到底部。然后, 在将焦点移向相反方向 (即, 从底部到原子核顶部) 时, 重新计算同一个原子核, 以确定 DAPI 体的计数数。</li>
		</ol>
</li>
		<li>在每一个稳定的雌雄同体中, 在两个性腺中的每一个至少十个睾丸中获得最成熟的未受精卵母细胞。野生型卵母细胞平均有 6 DAPI 体, 5 由于常染色体对, 和性染色体对。在稳定的 DAPI 卵母细胞中存在着12具尸体, 这表明这种菌株中的动物要么是部分 (4 组染色体, 3 x 染色体), 要么是完整的 (4 组染色体, 4 x 染色体) tetraploids。</li>
		<li>计算每株多 (至少 10) 卵母细胞的平均 DAPI 体数。有些染色体对会相互接触或右上方, 因此卵母细胞中 DAPI 体的数量通常小于染色体对的实际数量。</li>
		<li>选)进一步验证稳定的 DAPI 体的平均数量为 12, 以测试它们是否是完整的 (4A, 4X) 或部分 (4A, 3X) tetraploids 通过免疫荧光染色的染色体轴蛋白, 如 HTP-332。这种染色将区分染色体对通过显示一个十字模式的单一无关染色体存在的部分二、四倍体杂交。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

单倍体 (n) 配子的生产是在受精时生成一个双倍 (2n) 受精卵的关键。基因组的这种减少是在减数分裂过程中完成的, 在单个基因组重复后连续两个细胞分裂。为了生成单倍体配子,线虫与大多数其他后生一样, 在第一个分裂中隔离母系和父系衍生同系物, 而姐妹条染色单体从每个同源隔离在第二个分裂。在自然界中, 整个基因组多倍体产生的一个方法是, 在减数分裂过程中, 配子的生成不到一半的基因组大小。
50年来, 二、四倍体杂交的线虫线虫是可行和肥沃的。Nigon23和更高版本的 Madl 和赫尔曼22, 生成并识别了一小部分C. 线虫tetraploids, 其方法是通过热休克处理和使用遗传标记分别干扰减数分裂染色体分离。在30年后的24中, 使用此协议导出了另外一个briggsae二、四倍体杂交应变。这些 tetraploids 被用来调查线虫如何决定是男性还是雌雄同体, 倍性如何调节生长和大小, 以及分析减数分裂中的配对和联会25,26,38,39,40. 然而, 这些和其他需要使用特定 tetraploids 或无菌菌株的研究受到这种方法产生二、四倍体杂交菌株的困难的限制。
这里描述的协议允许生成稳定的完整的 4A, 4X 和部分 4A, 3X 二、四倍体杂交秀丽线虫菌株从任何初始的双倍的遗传背景或核型, 而不使用遗传标记。
Tetraploidy 中可能出现多个机制: 
Madl 和赫尔曼建议, 他们产生的二、四倍体杂交菌株可能来自一个无菌的中间状态。他们的菌株是通过多种世代的选择获得的, 或者是通过假定的无菌中间体与双倍雄性22。在rec-8突变体中染色体分割的缺陷导致了倍卵母细胞和精子的出现, 这表明了另一种可能的机制, 二、四倍体杂交动物可能会产生与rec-8 rna 干扰方案27。
rec-8对雌雄同体进行的 rna 干扰可以产生双倍卵母细胞和精母细胞, 这将导致二、四倍体杂交动物受精。与这种可能性相一致的是, 一些克隆的 F2 雌雄同体在下一代产生了稳定的离子株, 这表明克隆的 F2 雌雄同体已经二、四倍体杂交。在交叉方案中 , 第一代rec - 8对雌雄同体进行的 rna 干扰与未经处理的雄叉。倍精母细胞可能仍然出现在男性, 因为十字架是在存在的细菌表达rec-8 dsRNA, 因此, 男性暴露在rec-8 在交配期间至少3天的 rna 干扰。因此, 交叉施肥方案中的多倍体也可以由双精子卵母细胞受精形成。稳定的二、四倍体杂交菌株可能来自于两种配子之间的受精, 或从含有可变倍体卵母细胞的无精子动物与产生单倍体精子的双倍动物中产生。
重要考虑因素:
自我 vs 交叉施肥方案:
对 F1 雌雄同体进行自施肥的rec-8 rna 干扰方案, 并将治疗后的雌雄同体与未治疗的雄性杂交的方案均产生4A、4X 和4A、3X 二、四倍体杂交菌株。虽然多倍体最初与自施肥方案分离, 但更多的多倍体动物是无菌的, 因此这两种方案在生产二、四倍体杂交稳定菌株方面同样有效。在自我施肥计划中, 成功和不孕增加的原因仍然不得而知。虽然这两种方案都有同样的效率 , 但自施肥方案更容易 , 因为它不需要隔离雄配。此外, 当兴趣的应变是低效或有缺陷的交配, 自我施肥计划将是可取的。交叉施肥方案可用于生成包含两个以上版本的单一染色体的复杂 tetraploids。
修改和限制:
目前, 该协议涉及rec-8对rec-8基因表达 dsRNA 的细菌进行 rna 干扰处理。因此, 此协议在秀丽种中不起作用, 不响应通过喂食来进行 rna 干扰, 或者在组织41、42、43的环境或系统传播中出现缺陷的突变体。通过直接注入 dsRNA 直接向生殖中引入干扰处理, 可以解决这一问题。
无菌动物必须通过交叉二、四倍体杂交到从其派生的一个倍的动物生成, 因为这个方案不会产生稳定的无病毒菌株44, 45.只有15% 的卵播种由倍体孵化, 他们的后裔大多是无菌后裔由于体检。此外, 少数幸存的肥沃的后代往往是完整的或近 diploids 在几代人。这可能是, 至少部分, 因为卵母细胞部分矫正体通过分离第三个染色体进入极性身体在第一减数分裂分裂。
这个协议的一个不可逾越的限制是, 它不工作, 使 tetraploids 的突变体, 影响干扰机制的组成部分, 因为他们是抗干扰处理46。
诊断:
rec-8 rna 干扰:
一些考虑对于本议定书的成功至关重要。第一种方法是使用新鲜的 IPTG 和新鲜制作的 IPTG NMG 板, 在携带dsRNA (HT115) 克隆的细菌 rec-8 细菌中诱导rec-8 W02A2.6 生产。IPTG 是轻敏感的, 重要的是要减少光暴露于板材。IPTG 板可以储存多达一个月在4摄氏度在黑暗中。其次, rec-8 (W02A2.6) 从 Ahringer 库 (卡马斯和 Ahringer 2003) 中对细菌 HT115 菌株进行 rna 干扰, 产生了比其他可用rec-8 rec-8 克隆更强的HT115表型。
二、四倍体杂交应变维护:
二、四倍体杂交菌株的生长速度非常缓慢, 每代50个子代的产量为47。所有确定的二、四倍体杂交菌株都相对稳定, 但他们可以打破, 并成为双倍时, 强调 (乔纳森霍奇金个人沟通和我们未发表的意见)。因此, 重要的是要注意的是, 当二、四倍体杂交菌株生长在25摄氏度, 热休克, 饥饿, 或冷冻和解冻, 他们可以迅速恢复到 diploidy, 所以重要的是继续选择, 当解冻这些菌株或当使这些菌株暴露在压力下, 可能导致它们恢复。
可能的应用程序:
对全基因组多倍体化在细胞进化、细胞周期、基因表达和发育过程中的作用的调查依赖于: 一个有机体中含有不同倍体的细胞, 同一细胞与不同倍性或已经历进化最近的多倍体化事件和物理隔离的物种密切相关的物种类型 3, 5, 6, 7, 8 、11、18、19、20、48、49、50。虽然 tetraploids 可以从斑马鱼和老鼠模型系统中获得, 但它们的后代是无菌或 embryonically 致命的16,17。此外, 这些模型系统的寿命周期较C. 线虫,和可用的方法来产生多倍体动物是复杂和低效的.因此, 这种方法派生的线虫菌株将有助于进一步研究全基因组多倍体在多细胞有机体中的作用和作用。
由于一个二、四倍体杂交可以通过交叉二、四倍体杂交到原始的倍体来获得, 所以 diploids、倍体和 tetraploids 之间的比较在基因组拷贝的数量上是不同的, 它提供了一个独特和空前的机会来评估具有不同基因组大小 (或基因剂量) 的等效动物/器官/细胞。这一方案的灵活性和易用性使我们能够从不同的基因倍体背景或核型中产生许多二、四倍体杂交菌株。在减数分裂过程中, 有些菌株已经被用来查询同源染色体配对和联会的机制27。
携带荧光标记的野生型和突变二、四倍体杂交菌株将为了解基因组大小与核/细胞质比对细胞/细胞/器官和整个动物尺度、整个基因组之间的关系提供新的探索途径。多倍体化对适应, 形态, 基因剂量和表达, 组织和器官的发展。除了生物尺度的研究外, 二、四倍体杂交菌株的生成还将对与细胞外信号、基因组不稳定性、endoreduplication、整个基因组相关的基本生物学问题有进一步的质疑。重复, 基因剂量, 适应压力, 发展抗药性药物, 以及机制的形态。

整个基因组多倍体存在于自然界中, 通常是适应、形态、器官、创伤愈合和生物结垢的必要步骤;它也知道促进癌症和抗药性的药物1,2,3,4,5,6,7,8,9,10,11,12. 农业和渔业工业通过化学处理产生多倍体植物、鱼类和贝类 (例如、秋水仙碱和 orzalin) 以获得更快的生长速度和笨重的农作物和牲畜13, 14,15。实验性和低效生产的 tetraploids 存在的鼠标和斑马鱼模型系统16,17。然而, 大多数或所有多倍体多细胞动物所产生的 embryonically 致命或无菌, 因此不理想的实验室研究的影响, 多倍体的细胞生物。因此, 对多细胞生物体中的整个基因组多倍体化的任何理解都限于与最近的进化多倍体化事件密切相关的物种18, 19, 20.多倍体化的生物学作用或后果的查询的路径是使用线虫模型系统. 重要的是, C. 线虫是一种易于处理的遗传系统, 通常作为一个倍体存在, 仅包含五染色体 (a) 和每个基因组的一性染色体 (X), 是透明的, 允许体内观察生物过程, 并生命周期短 3-4 天 (从卵子到性成熟成人)。C. 线虫已被证明可以繁殖为二、四倍体杂交, 这是最常见的类型的整个基因组多倍体的性质。tetraploids 与 diploids 杂交, 可产生无菌动物, 但其倍性不稳定, 两代内菌株就成了双倍。
在过去的几十年中, 只有少数的可行和肥沃的线虫tetraploids 菌株被隔离在实验室中, 使用的策略是费力的, 只产生有限类型的菌株21,22, 23. 此策略生成C. 线虫tetraploids 的热休克治疗, 这可能会影响配子中的染色体分离, 其次是对假定的多倍体动物使用遗传标记进行筛选。这些 tetraploids 是非常有用的, 在询问如何这个线虫决定是否成为男性或雌雄同体。后来的研究使用可用的菌株来调查在减数分裂期间联会的生长、基因剂量和调控, 此线虫21,24,25,26。不幸的是, 这些研究由于难以产生新的二、四倍体杂交菌株和这些菌株所含的背景基因标记而受到限制。这里显示的是一个简单而快速的协议来生成稳定的 tetraploids, 它已经被用来产生菌株来研究联会在减数分裂过程中的调节27。
与自然界一样, 多倍体化的形成是由双倍而不是单倍体配子产生的。我们发现, 一个减数分裂特定的内聚力联合体组件突变体 rec-8 生成双精子和卵母细胞表明, 击倒 rec-8 基因将导致二、四倍体杂交后代的生产 (图 1) 27, 28,29。生成二、四倍体杂交菌株只需用 RNA 干涉 (rna 干扰) 敲掉rec-8 两代, 以phenocopy rec-8 变种人的配子。假定的多倍体可以很容易地识别其长于正常体型。多倍体被确认为完全或部分 tetraploids, 通过计数每核的染色体的数量, 一旦稳定的线建立。
此处描述的策略允许在不使用遗传标记的情况下, 从任何初始的双倍遗传背景或核型中生成稳定的二、四倍体杂交秀丽线虫菌株。由于该协议比以前使用的方案更高效、更通用、更简单, 因此它将扩展用于查询多倍体化在发展、基因组稳定性和多细胞进化等基本过程中的作用所需的工具。生物.在使用本议定书时, 唯一可预见的限制是在遗传背景下抗 rna 干扰。

<table>
	<tbody>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Motic Microscopy</td>
			<td>SMZ171</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>NGM agar plates</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>60 mm x 15 mm Petri Dishes, Sterile</td>
			<td>Tritech Research</td>
			<td>T3305</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 15 mm Petri Dishes, Sterile</td>
			<td>Tritech Research</td>
			<td>T3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological Agar, 5 kg</td>
			<td>VWR</td>
			<td>89140-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride, Biotechnology Grade</td>
			<td>VWR</td>
			<td>97061-278</td>
			<td></td>
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		<tr>
			<td>Peptone, BD Bacto</td>
			<td>VWR</td>
			<td>90000-368</td>
			<td></td>
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			<td>Ampicillin Trihydrate</td>
			<td>VWR</td>
			<td>97062-300</td>
			<td></td>
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			<td>IPTG, dioxane-free</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Growth Culture</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>LB Lennox Broth</td>
			<td>IBI Scientific</td>
			<td>89126-176</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LB Agar plates</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>LB Agar Lennox</td>
			<td>IBI Scientific</td>
			<td>89126-182</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Ampicillin Trihydrate</td>
			<td>VWR</td>
			<td>97062-300</td>
			<td></td>
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			<td>Tetracycline Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP912-100</td>
			<td></td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Hoechst 33258, Pentahydrate</td>
			<td>Biotium</td>
			<td>40045</td>
			<td></td>
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			<td>DAPI</td>
			<td>Biotium</td>
			<td>40011</td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>Ethanol Fixation</td>
			<td></td>
			<td></td>
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			<td>Ethanol, Pure, 190 Proof (95%), USP</td>
			<td>Koptec, VWR</td>
			<td>89125-166</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>M9 Buffer</td>
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			<td></td>
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			<td>Sodium chloride, Biotechnology Grade</td>
			<td>VWR</td>
			<td>97061-278</td>
			<td></td>
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			<td>Potassium phosphate, Monobasic Anhydrous Grade</td>
			<td>VWR</td>
			<td>97062-346</td>
			<td></td>
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		<tr>
			<td>Sodium phosphate, monobasic dihydrate</td>
			<td>Fisher Scientific</td>
			<td>AC271750025</td>
			<td></td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>RNAi Clones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HT115 bacteria expressing rec-8 dsRNA</td>
			<td>Source Bioscience</td>
			<td>W02A2.6</td>
			<td></td>
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			<td>HT115 bacteria expressing rec-8 dsRNA</td>
			<td>Dharmacon</td>
			<td>RCE1182-202299820</td>
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manipulation of ploidy in caenorhabditis elegans

涉及全基因组多倍体的机制在发展和进化中起着重要作用;此外, 二、四倍体杂交细胞的异常生成也与癌症的进展和耐药性的发展有关。到目前为止, 在不产生大部分不孕后代的情况下, 很容易操控多细胞动物的倍性是不可行的。这里提出了一个简单快速的协议, 以生成二、四倍体杂交秀丽线虫动物从任何双倍菌株。该方法允许用户在减数分裂过程中产生染色体分离的偏倚, 最终增加C. 线虫的倍性。该策略依赖于瞬时减少rec-8基因的表达, 以生成双倍配子。一个rec-8突变体产生双倍配子, 在受精时可能产生 tetraploids。这种可驯服的方案已被用来产生二、四倍体杂交菌株携带突变和染色体重排, 以获得洞察的染色体动力学和相互作用的配对和联会的减数分裂。该方法有效地生成了无遗传标记的稳定二、四倍体杂交菌株, 可应用于任何双倍菌株, 可用于提取无菌的 C. 线虫。这种直截了当的方法对于研究与基因组不稳定性、基因剂量、生物尺度、细胞外信号、适应压力、抗药物的发展等相关的基本生物学问题是有用的,形态的机制。

rec-8减数分裂内聚力联合体成分函数的损伤导致了倍性配子:
内聚力联合体成分突变体减数分裂分裂的影像学研究 rec-8 精子和卵母细胞揭示了生成二、四倍体杂交动物的可能机制 (图 1)27 ,29,33。机械化, rec-8突变体精子和卵母细胞的减数分裂分化缺陷不同;然而, 雄性和雌性双配子是由rec-8突变体产生的。
在野生类型减数分裂中, 同源染色体通过交叉重组暂时连接到另一个, 并以单位或二价 (图 1A)34的形式进入第一个减数分裂分裂。在第一个分裂期间, 同源染色体 (同系物) 彼此隔离, 而姐妹条染色单体在每个同源保持在一起, 直到第二个分裂。虽然染色体分离的模式是相同的雌性和男性配子, 卵母细胞分裂是不对称的, 而精分裂是对称的, 经过专门的胞质分裂。在每个分裂, 卵母细胞放弃一半的分裂产品成一个小的极性身体。因此, 每一个卵母细胞的前驱体都产生一个单倍体卵母细胞和两个极性体 (图 1B, C)35。反之, 单个精前驱体通过两个对称分裂产生四功能精子。第二个分裂高潮与四精子从残余的身体萌芽。此胞质分裂是特殊的, 因为精子的模式和数目由中心体36决定。
在rec-8突变配子的前体中, 同源染色体之间的交叉重组不发生, 同系物在第一次减数分裂的起始处29,34开始时没有连接为二价。每个同源的姐妹条染色单体分开在第一个分裂, 而不是保持在一起, 直到第二个分裂, 就像他们在野生类型配子。有趣的是, 在第二个分裂期间的rec-8突变表型不同于雄性和雌性配子, 因为卵母细胞无法接受胞质分裂而精母细胞经历了相对正常的胞质分裂(图 1B-F)27,28,29. 双卵母细胞的出现是因为在第二个分裂中, 它们既不能分离染色体又不胞质分裂, 因此它们不会挤压第二个极性体(图 1B, C).精母细胞在rec-8 germlines 中进行精子发芽或胞质分裂, 但通常将这两套染色体与精子中的一组分离, 以产生双倍精子和缺少染色质的精子(图1D-F)27。图 1F显示了缺乏染色质的rec-8精子比例的量化。
在rec-8突变体中, 雌性和雄性倍性配子的形成表明, 这种突变型可以潜在地用于产生全基因组二、四倍体杂交菌株。
生成二、四倍体杂交 C . 线虫菌株:
在所有生成的二、四倍体杂交菌株中,在rec-8 基因中存在突变, 可以通过rna29、37瞬态击倒rec-8 来避免。这假定通过 rna 干扰减少rec-8函数会产生可能导致整个基因组二、四倍体杂交动物的双倍配子,作为rec-8 变种人做29, 37.这项协议的关键是, rec-8突变体都产生了双配子和陛下相当多的年轻, 相反的其他减数分裂突变体, 这导致异倍体配子, 主要是无菌或胚胎/幼虫致死。
通过使用两种策略之一 (参见协议、图 2和表 1)27, 通过喂养C. 线虫细菌表达rec-8 dsRNA 生成多个二、四倍体杂交菌株。Tetraploids 可以由自我施肥雌雄同体产生三代的培养皿与细菌表达rec-8 dsRNA。通过这个策略, 一个雌雄同体被放置在新鲜制作的rec-8 rna 干扰板和它的后代被转移几天后到新的板块与新鲜诱导的rec-8 rna 表达细菌 (见协议)。此外, tetraploids 可以通过跨越第一代雌雄同体喂养的细菌表达rec-8 dsRNA 与未经处理的雄性基因型在培养皿中的细菌, 表达rec-8 dsRNA (图 2)。在这种情况下, 在交配过程中, 十字架上的雄性暴露于从 L4 阶段开始的rec-8 rna 干扰细菌。这两种方案都产生了二、四倍体杂交动物27。表 1显示了使用此处介绍的方案获得的二、四倍体杂交菌株。无菌和二、四倍体杂交线虫的大小比 diploids 大, 但无菌菌株是不稳定的, 并倾向于在一、两代内成为双倍, 而二、四倍体杂交菌株是相对稳定的 22, 23.假定的二、四倍体杂交菌株被确定为大于原来的应变 (雌雄同体), 播种仅是离子后代(图 3A)。隆动物很容易辨认-野生型加倍动物是 tetraploids 的长度的二三, 并且他们的身体不做一个额外的弯曲, 在它的向前正弦运动可能被注意 (图 3A)。
二、四倍体杂交菌株在二、四倍体杂交雌雄同体卵母细胞中存在12条染色体对, 与双雌雄同体卵母细胞的六染色体对(图 3B、 C和电影 1) 相比较。
二、四倍体杂交类:
两种类型的二、四倍体杂交雌雄同体被确定: 一播种男性在相似的频率作为双倍雌雄同体和其他播种男性在更高的频率(表 1)。这两种二、四倍体杂交雌雄同体的不同之处在于, 与双雄染色体相似的类产生频率是二、四倍体杂交的, 而产生高频率的雄性的类则是雌雄同体是二、四倍体杂交的染色体, 但对性别染色体的无细胞性 (4, 3X)。后一类的 tetraploids 稳定, 生产 4A, 3X 雌雄同体和 4A, 2X 男性。
二、四倍体杂交菌株的生长速度较慢, 并且产生的巢粒大小与它们派生的 diploids 相对应, 就像以前的方法22 23 所产生的应变一样.Madl 和赫尔曼22建议, 二、四倍体杂交菌株中死胚的比例增加可能是由于卵母细胞中的体检, 然而对二、四倍体杂交菌株的表面检查并未显示出足够高的异倍体数量。卵母细胞或不正常的卵母细胞或精分裂为观察到的巢大小的减少 (影片 2和图 3C, D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57296/57296fig1.jpg"> 图 1: rec-8突变体中的配子暗示了产生稳定多倍体污渍的可能机制.(A) 野生类型和rec-8突变减数分裂分裂的染色体组织和隔离模式示意图。在野生类型减数分裂中, 同源染色体分离在第一减数分裂分裂。姐妹条染色单体在每个同源朝向同一个纺锤杆并且保持在一起直到第二个分裂。在rec-8突变体中, 同系物不形成分频, 因此没有连接。与野生类型不同的是, rec-8姐妹条染色单体在第一个减数分裂分裂中远离彼此并分离。(B) 显示雌性原核和挤压极性体的野生类型和突变卵母细胞图 (不描述雄性原核)。在野生型卵母细胞中, 两个非对称减数分裂分裂导致两个极性体的挤压。然而, 在rec-8突变体中, 第二个极性身体挤压失败导致了双倍卵母细胞。(C) 野生类型和rec-8突变卵母细胞的图像, 表达 mCherry:: 组蛋白 H2B。箭头表示野生型卵母细胞中的两个极性体, 另一个位于rec-8变种中。缩放栏为5µm. (D和E) 精子的实时图像, 从野生类型和rec-8变种动物表达 mCherry:: 组蛋白 H2B (洋红)。箭头表示 anucleate 精子。缩放条为2µm (D) 精母细胞在野生类型和rec-8 突变体中进行第二个 (萌芽) 分裂。野生型精母细胞经过对称分裂, 导致四芽精子, 每一个都有单倍体染色体补体。在rec-8突变体精母细胞中, 第二个分裂中的染色体分离被削弱。大多数情况下, 在第二次减数分裂分裂中, 一大团染色质残留在残余体 (RB) 或两个姐妹精子中的一个。这就产生了 anucleate rec-8突变精子 (由箭头表示) 或双倍精子。(E) 在野生类型和rec-8突变体中, 使用差分干涉对比 (DIC) 和荧光显微镜可视化后萌芽精子的实时图像。野生型精子均有类似的染色单体质量。rec-8突变体精子形成 anucleate 精子。(F) 对野生类型和rec-8突变体 anucleate 精子进行量化。rec-8突变体产生了38.5% 的 anucleate 精子, 而野生型则少于1.6%。费舍尔的确切测试表明, 与野生类型相比, rec-8突变体 anucleate 精子 (p ≤0.0001) 的发病率明显较高。误差线表示标准偏差。<a href="//cloudflare.jove.com/files/ftp_upload/57296/57296fig1large.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/57296/57296fig2.jpg"> 图 2: 从任何应变中生成和隔离二、四倍体杂交 C . 线虫的方案。右侧的箭头表示协议的时间线, 从1天开始, 进展到17天。同样的结果也可以通过在9天将雌雄同体到未治疗的男性身上来实现。括号将步骤的描述连接到时间线。未经处理的动物是橙色的, 被对待的动物是红色的, 更大的动物在大小, rec-8 dsRNA 表达细菌被描述作为透明的板材以透明红色背景和规则 OP50 细菌被描述在透明灰色背景板。程序是在15°c, 除非另有说明。<a href="//cloudflare.jove.com/files/ftp_upload/57296/57296fig2large.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/57296/57296fig3v2.jpg"> 图 3: 二、四倍体杂交示例。(A) 二、四倍体杂交 (MSC2) 动物的明亮场图像和它从 (AV740) 派生的双倍应变。二、四倍体杂交 C . 线虫总体上比双倍的大, 更长。这种体型的差异会导致运动二、四倍体杂交体的另一正弦曲线, 可作为二、四倍体杂交衍生物的筛选标准。缩放条是0.1 毫米 (B, C) 荧光图像的双倍 (AV740) 和二、四倍体杂交 (MSC1) 最成熟的未受精卵母细胞 nulcei, 在减数分裂分裂之前。二次筛查是通过观察已建立的离子菌株未受精卵母细胞的染色体对的数量, 如影片 1中所示的 MSC1 菌株表达 Mcherry::H2B 和 GFP:: 蛋白在生殖。缩放条为5µm. (D) 从二、四倍体杂交卵母细胞减数分裂分裂的时间间隔(电影 2) 中描绘一般正常减数分裂的图像。箭头头标记极性身体和一条虚线标记卵母细胞的皮层在蝗受精囊和被精子包围;时间在几分钟内失效。刻度条为10µm.<a href="//cloudflare.jove.com/files/ftp_upload/57296/57296fig3v2large.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>基因</td>
			<td>二、四倍体杂交衍生物 (染色体的集合: X 染色体的 #) *</td>
			<td>家长应变 双倍)</td>
		</tr>
<tr>
<td>meIs16 [pie-1p::mCherry::his-58 + unc-119 (+)];</td>
			<td>MSC1 (4 a: 4 x)</td>
			<td rowspan="6">MSC0</td>
		</tr>
<tr>
<td>
ruIs57 [pie-1p::GFP:b-蛋白 + unc-119 (+)]
</td>
			<td>MSC2 (4 a: 3 x)</td>
		</tr>
<tr>
<td></td>
			<td>MSC3 (4 a: 4 x)</td>
		</tr>
<tr>
<td></td>
			<td>MSC5 (4 a: 3 x)</td>
		</tr>
<tr>
<td></td>
			<td>MSC6 (4 a: 4 x)</td>
		</tr>
<tr>
<td></td>
			<td>MSC8 (4 a: 4 x)</td>
		</tr>
<tr>
<td rowspan="3">unc-119 (ed3) III;ddIs6;ddIs6 [tbg-1:: GFP + unc-119 (+)];ltIs37;ltIs37 [pAA64; pie-1p::mCherry::HIS-58 + unc-119 (+)] IV</td>
			<td>MSC14 (4 a: 3 x)</td>
			<td rowspan="3">TMR17</td>
		</tr>
<tr>
<td>MSC15 (4 a: 3 x)</td>
		</tr>
<tr>
<td>MSC16 (4 a: 4 x)</td>
		</tr>
<tr>
<td>11 (me44)/nT1 IV;+/nT1 [qIs51 [肌-2:: gfp Ppes-10::gfp, PF22B7.9:: gfp]] V</td>
			<td>AV800&
</td>
			<td>AV776</td>
		</tr>
<tr>
<td>meIs8 [pie-1p::gfp::cosa-1 + unc-119 (+)] II;</td>
			<td rowspan="3">AV809&
</td>
			<td rowspan="3">AV727</td>
		</tr>
<tr>
<td>ltIs37 [pie-1p::mCherry::his-58 + unc-119 (+)] IV;</td>
		</tr>
<tr>
<td>ltIs38 [pie-1p::gfp::p h (PLC1delta1) + unc-119 (+)]</td>
		</tr>
<tr>
<td>meIs8 [pie-1p::gfp::cosa-1 + unc-119 (+)] II;mnT12 (X;四)</td>
			<td>AV826&
</td>
			<td>AV695</td>
		</tr>
<tr>
<td>mIn1 [dpy-10 (e128) mIs14 [肌-2:: gfp pes-10::gfp]]/</td>
			<td rowspan="2">AV810&
</td>
			<td rowspan="2">DR2078</td>
		</tr>
<tr>
<td>
bli-2 (e768) unc-4 (e120) 二 </td>
		</tr>
<tr>
<td rowspan="2">
ruIs32 [pie-1::GFP::H2B + unc-119 (+)] III </td>
			<td>AV822&
</td>
			<td rowspan="2">AZ212</td>
		</tr>
<tr>
<td>AV823&
</td>
		</tr>
<tr>
<td>C. briggsae-mfIs42-sid-2 + Cel-myo-2::D sred]</td>
			<td>AV824&
</td>
			<td>JU1018</td>
		</tr>
</tbody></table>表 1: 生成的二、四倍体杂交菌株。* 在减数分裂分裂之前, 除了雄性播种的比例外, 通过在卵母细胞中评分价体 (连接同源对) 和 univalents (个体同系物) 的数量推断。
<img alt="Movie 1" src="/files/ftp_upload/57296/57296video1.jpg"> 影片 1: 筛选以确认稳定的 tetraploids 是否是完全或部分的.电影从一个野生型雌雄同体的图开始, 突出区域成像 (未受精的卵母细胞), 通过计数染色体对来识别 tetraploids。在图之后, 一系列的 Z 堆叠影片和投射显示了二、四倍体杂交动物的性腺和 DAPI 染色的或活的图像, 表达 Mcherry::H2B 组蛋白的菌株。通过计数未受精卵母细胞中连接的同源染色体对的数量进行筛选。计数的 MCherry 或 DAPI 染色体是在未受精的卵母细胞, 最接近精子储存蝗受精囊 ("-1 卵母细胞")。染色体在这个卵母细胞是最凝聚和分离, 允许更准确的同源对计数。每株10多只动物进行筛查, 以确保-1 卵母细胞染色体计数准确。堆叠厚度为0.2 µm.<a href="http://cloudflare.jove.com/files/ftp_upload/57296/Movie1.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Movie 2" src="/files/ftp_upload/57296/57296video2.jpg"> 影片 2: 二、四倍体杂交卵母细胞分裂看起来正常。表达 Mcherry::H2B 组蛋白和 GFP 的二、四倍体杂交菌株卵母细胞分裂的时间间隔: β-蛋白。分裂的时间和模式看起来很正常。每2.5 分钟拍摄一次图像<a href="http://cloudflare.jove.com/files/ftp_upload/57296/Movie2.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>

Watch this Scientific Journal Video about Manipulation of Ploidy in Caenorhabditis elegans at JoVE.com

Manipulation of Ploidy in Caenorhabditis elegans

这种方法允许生成二、四倍体杂交和无菌的秀丽线虫从任何双倍菌株。这种方法产生的多倍体菌株用于研究减数分裂前期的染色体相互作用, 这一方法对于研究细胞、发育、进化和肿瘤生物学等重要的基本问题是有用的。

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Genetics

Manipulation of Ploidy in Caenorhabditis elegans

10.9K 次观看.  City University of New York. 这种方法允许生成二、四倍体杂交和无菌的秀丽线虫从任何双倍菌株。这种方法产生的多倍体菌株用于研究减数分裂前期的染色体相互作用, 这一方法对于研究细胞、发育、进化和肿瘤生物学等重要的基本问题是有用的。

视频: Manipulation of Ploidy in Caenorhabditis elegans

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Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

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Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

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Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

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Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

微卫星DNA基因分型和流细胞学比较分析形式固定石蜡嵌入水合体摩尔组织

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...