Name: 作者聚焦：PGC 冷冻保存 – 一种长期保存果蝇菌株的替代方法
Uploaded: 2023-12-01T12:20:00
Duration: 1 min 13 s
Description: Watch this Scientific Journal Video about PGC Cryopreservation – An Alternative Approach Towards Long-Term Preservation of Drosophila Strains at JoVE.com

我们感谢京都果蝇库存中心的苍蝇菌株。我们还要感谢 Wanda Miyata 女士对手稿的英文编辑，以及 Edanz 的 Jeremy Allen 博士（https://jp.edanz.com/ac 年）编辑了这份手稿的草稿。这项工作得到了日本医学研究与开发机构 （AMED） 对 T.T.-S.-K. 的赠款（JP16km0210072、JP17km0210146、JP18km0210146）的支持，AMED 对 SK 的赠款（JP16km0210073、JP17km0210147、JP18km0210145）、AMED 对 T.T.-S.-K 的赠款（JP20km0210172）。S.K.，日本科学振兴会（JSPS）向T.T.-S.-K.提供的科学研究补助金（C）（JP19K06780），以及JSPS向S.K.提供的创新领域科学研究补助金（JP18H05552）。

1.设备准备
<ol><li>显微操纵系统：组装显微操纵系统以收集和移植细胞（图1A）。</li>
	<li>PGC收集载玻片（图2A）<ol><li>要制备庚烷胶，请剪下约 30 厘米长的双面胶带，并将其浸泡在 7 mL 工业（常规）级庚烷溶液中过夜。</li>
		<li>在载玻片背面绘制两条平行的参考线，用于胚胎对齐。</li>
		<li>使用巴斯德移液管将上述庚烷胶滴在载玻片上（在没有线条的一侧）。风干载玻片表面，直至变白。</li>
		<li>重复庚烷胶滴的添加和扩散，并再次干燥载玻片。 注意： 胶水可防止液体溶液在平面上扩散，并更容易将水溶液装入针头中。</li>
		<li>要制作胚池框架，请在砧板上粘贴三层 0.2 毫米厚的标准乙烯基胶带，例如电工胶带。将胶带切成 1.5 厘米宽的矩形。然后，剪掉所有三层胶带，留下一个 2 到 3 毫米的框架。 注意：在对齐胚胎后贴上胚池框架，以形成胚胎池。</li>
	</ol></li>
	<li>移植针 注意：在本研究时，所有市售针头对于 PGC 冷冻保存来说都太窄或太宽。<ol><li>使用玻璃毛细管和拉拔器制作针头。我们使用 NARISHIGE PN-31 拉拔器，加热器电平为 85.0-98.4，磁铁主电平为 57.8，磁铁子电平为 45.0。</li>
		<li>要制造壁厚约为 1 μm 且尖端长度约为 200 μm、内径为 10-12 μm 的针头，请按照以下三步工艺抛光针尖（图 3）。首先，以 30 rpm 的速度以 780° 角向下研磨针尖，直到针尖的内径为 10-12 μm。第一个研磨步骤大约需要 1 小时。 注意： 为避免折断针尖，请先旋转磨石，然后轻轻地将针向下移动到磨石上。</li>
		<li>在针的顶部画一条线以跟踪所需的角度。将针头逆时针旋转 90°，然后以 180 rpm 的速度再次抛光。这大约需要 5 分钟。</li>
		<li>将针头顺时针旋转 45°，然后以 180 rpm 的速度抛光一秒钟。</li>
		<li>将装有一滴铬酸混合物（注意：有毒）的收集载玻片放在显微镜载物台上。将针头以相对于载玻片表面10°-13°的角度连接到毛细管支架（图1D），小心地向下移动针头，并将针头浸入铬酸混合物中。</li>
		<li>通过拉动和推动柱塞（图1B），机械地将溶液从针头中加载和排出数次，以去除针头中的玻璃碎屑。一定要清洁外墙。</li>
		<li>用蒸馏水清洗针头的内侧和外侧两次，以彻底去除铬酸。</li>
	</ol></li>
</ol>2. PGCs的采集和冷冻保存
<ol><li>收集胚胎<ol><li>将适当数量的目标供体菌株的果蝇（胚胎收集杯的每个性别约450只）转移到带有胚胎收集板的胚胎收集杯中（图1E），并在25°C下孵育。 我们通常使用3至5日龄的亲蝇，这些苍蝇在室温（23-25°C）下在不那么拥挤的条件下饲养。</li>
		<li>进行两次 30 分钟的预收集并丢弃任何产卵。由于雌性可以保留在输卵管中发育的受精卵，因此该步骤是步骤2.1.3中同步产卵所必需的（图4）。</li>
		<li>在两次预收集后，收集胚胎50分钟，然后将收集的胚胎在25°C的加湿室中孵育，以使胚胎发育到胚层阶段（早期阶段5,17）。孵育时间通常为100分钟，但可以延长至120分钟，具体取决于菌株（图4）。 注意： 加湿室是通过在塑料盒底部放置湿纸巾并在使用前喷洒水雾制成的。在早期 5 期胚胎中，PGC 形成完成，但体细胞化尚未完成。在步骤2.4中，在复合显微镜下确定胚胎的确切阶段。</li>
	</ol></li>
	<li>胚胎去瘤<ol><li>将一滴蒸馏水沉积在不锈钢网过滤器（150目，109μm开口，60μm线径; 图1F）。使用镊子，从胚胎收集板中收集胚胎并将它们放入水滴中。</li>
		<li>将纸巾从下方压在过滤器上以吸收水分。将新鲜的5%（Cl）次氯酸钠溶液滴入胚胎中，并连续敲击过滤器10秒。</li>
		<li>直接用蒸馏水泼洒胚胎来清洗胚胎，然后从下面将纸巾压在过滤器上以吸收水分。重复此步骤 3 次。</li>
	</ol></li>
	<li>对齐去皮胚胎<ol><li>在体视显微镜下，使用镊子移植胚胎。沿着两条参考线在PGC收集载玻片上将去角质的胚胎排成两排（图2A）。胚胎的方向是它们的前部向右（要操作的一侧），腹侧朝上。 注意：此步骤应在 20 分钟内完成，在此期间我们通常对齐大约 40 个胚胎。</li>
		<li>在PGC收集载玻片上的胚胎周围贴上胚池框架。将1μLCPA溶液（1xEphrussi-Beadle Ringer溶液，EBR，含有20%乙二醇和1M蔗糖;1x EBR：130mM NaCl，5mM KCl，2mM CaCl2和10mM Hepes，pH 6.9）滴在框架封闭区域的两个不同位置，并用硅油填充池以防止胚胎变干（图2A）。 注：要制备 CPA 溶液，将 10.26 g 蔗糖完全溶解在含有 3 mL 10 x EBR 溶液的约 20 mL 蒸馏H 2O 中。加入 6 mL 乙二醇，然后加入蒸馏 H2O 至 30 mL。充分混合后，通过0.22毫米一次性膜过滤溶液。</li>
	</ol></li>
	<li>收集 PGC<ol><li>将步骤2.3.2的PGC收集载玻片放在配备显微操纵器系统的显微镜的载物台上。将针头连接到毛细管支架上，并将左排中的第一个胚胎和针尖带入同一焦平面。将硅油放入针中 2-3 秒。</li>
		<li>从左排的胚胎开始PGC收集。使用20倍物镜，轻轻地将针尖移动到胚胎前端的表面，然后将胚胎穿透到后端，不是通过移动针头，而是通过移动显微镜载物台。</li>
		<li>当针尖到达后端时，稍微缩回针头，将针头中的任何蛋黄完全排出体细胞层内。</li>
		<li>在保持针头压力恒定的同时，将针尖移动到后极内的 PGC，然后轻轻但不要花费太多时间加载 PGC。</li>
		<li>快速将针头从胚胎中拉出，将针头中的蛋黄和其他污染物排入硅油池中，将PGC保持在针头中。然后，从池中装入干净的硅油。</li>
		<li>对左行中的其他胚胎重复步骤2.4.2至2.4.5。在从新胚胎中收集PGC之前，将步骤2.4.5中加载的硅油尽可能多地沉积在体细胞层内，同时将加载的PGC保持在针中。这确保了新加载的 PGC 与先前收集的 PGC 相邻，它们之间没有任何干预材料。</li>
		<li>从左排胚胎中完成PGC收集后，尽可能将PGC与蛋黄和其他污染物分离。为此，将针头中的所有PGC沉积到胚胎表面，并去除另一个相邻胚胎的任何蛋黄或其他污染物。</li>
		<li>接下来，从右排的胚胎中收集PGC。合并从右行和左行收集的 PGC。</li>
	</ol></li>
	<li>将冷冻保护剂 （CPA） 应用于 PGC<ol><li>用一滴 CPA 清洗针头后，将另一滴新鲜的 CPA 装入针头中，并将 CPA 添加到沉积在胚胎上的 PGC 中。CPA的数量应与PGC的数量相等。</li>
		<li>添加 CPA 后，从 PGC 集群 1-2 秒中删除尽可能多的 CPA。添加 CPA 后，PGC 会略微收缩并立即变成方形。</li>
		<li>清空针头，然后加入硅油 5 秒或更长时间。加载所有收集的 PGC，然后再次加载硅油 5 秒或更长时间。PGC现在夹在两层硅油之间（图2B）。 注意： 尽可能多地去除蛋黄、CPA 和其他污染物很重要。</li>
	</ol></li>
	<li>低温保存 PGC<ol><li>打开三通旋塞阀（图1C），然后将针头从显微操纵器上拆下。用软纸巾吸干针头表面的油。不要直接用纸巾接触针尖。</li>
		<li>将针头连接到针架上，并使用乙烯基胶带将其锁定在底座上（图1H）。在支架管上贴上标签。</li>
		<li>将针头朝下，将其浸入液氮中，快速冷冻支架。在液体停止从架子中嘶嘶作响之前，不要松开支架。</li>
		<li>将支架存放在液相区域的液氮储罐中，而不是气相区域。</li>
	</ol></li>
</ol>3. PGCs的解冻和移植
<ol><li>从非配子宿主果蝇中收集、去皮和比对胚胎<ol><li>按照步骤2从非配子宿主果蝇中收集和去胚胎去皮。</li>
		<li>将第5阶段的非配子宿主胚胎对齐在移植载玻片上。然而，这一次，将后部定向右侧（要操纵的一侧），腹侧定向顶部（图5）。在20分钟内将大约30个胚胎排成两排。</li>
		<li>在对齐胚胎时，如果室内湿度需要，请操作加湿器2-10分钟（表1）。理想的湿度为 30% 至 40%，但这可能因热条件而异。</li>
	</ol></li>
	<li>在宿主胚胎中解冻和移植PGC<ol><li>要快速解冻冷冻保存的PGC，将装有针头的支架滑入室温1x EBR溶液中，针头朝下，并保持浸没10秒。</li>
		<li>将移植载玻片放在显微镜的载物台上。将冻融的针头连接到毛细管支架上，并将左排中的第一个胚胎和针尖带入同一焦平面。</li>
		<li>使用20倍物镜，轻轻地将针尖移动到胚胎后端的表面。</li>
		<li>轻轻地戳每个胚胎的外部，确保它们慢慢恢复到原来的形状。刺激将确认胚胎的内部压力不会太高或太低。</li>
		<li>轻轻移动针头，从后极穿透胚胎。</li>
		<li>轻轻地将大约 10-20 个 PGC 沉积在后极内，正好在卵黄膜和胚胎的体细胞层之间。避免将它们沉积在体细胞层中。如果卵黄周围液从胚胎中渗出，将渗漏的液体吸入针头并将其取出。</li>
		<li>将针头从胚胎中缩回。对后续胚胎重复步骤3.2.5和3.2.6。</li>
	</ol></li>
</ol>4. 孵化胚胎和恢复供体菌株
<ol><li>去除任何未接受移植PGC的胚胎，并将剩余的胚胎在25°C的加湿室（图1G）中孵育。</li>
	<li>在移植后24小时或更长时间以及孵化后尽快，使用镊子将孵化的幼虫捡起并转移到标准 果蝇 食品瓶中，并在25°C下孵育。</li>
	<li>为了恢复该菌株，将新出现的雌性和雄性杂交（图6）。 注意：无配子宿主可以一次恢复整个基因组，而无需与平衡染色体菌株杂交。非配子雄性在小瓶中的共存无关紧要，因为雌性即使与它们交配，也不会表现出长期的交配后反应，包括对重新分配的接受度降低18,19。</li>
</ol>

用于果 蝇 菌株长期保存的 PGC 冷冻保存

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A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Drosophila+Embryogenesis.">Stages of Drosophila Embryogenesis.</a> The Embryonic Development of Drosophila. , (1997).</li><li>Manning, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sperm+factor+affecting+the+receptivity+of+Drosophila+melanogaster+females.">A sperm factor affecting the receptivity of Drosophila melanogaster females.</a> Nature. 194, 252-253 (1962).</li><li>Kubli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-peptides:+seminal+peptides+of+the+Drosophila+male.">Sex-peptides: seminal peptides of the Drosophila male.</a> Cell Mol Life Sci. 60, 1689-1704 (2003).</li><li>Lehmann, R., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+segmentation,+pole+cell+formation,+and+embryonic+polarity+require+the+localized+activity+of+oskar,+a+maternal+gene+in+drosophila.">Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in drosophila.</a> Cell. 47 (1), 141-152 (1986).</li><li>Kiger, A. A., Gigliotti, S., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+the+essential+Drosophila+Nucleoporin+nup154:+allelic+differences+due+to+an+outward-directed+promoter+in+the+P-element+3&#8242;+end.">Developmental genetics of the essential Drosophila Nucleoporin nup154: allelic differences due to an outward-directed promoter in the P-element 3&#8242; end.</a> Genetics. 153 (2), 799-812 (1999).</li><li>Rienzi, L. F., 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=Perspectives+in+gamete+and+embryo+cryopreservation.">Perspectives in gamete and embryo cryopreservation.</a> Semin Reprod Med. 36 (5), 253-264 (2018).</li></ol>

作者没有利益冲突需要声明。

PGC冷冻保存和恢复成功的关键因素是使用好的胚胎。应使用年轻雌性（例如，3 至 5 日龄）进行胚胎采集。供体和宿主胚胎都通过显微镜检查进行评估，并且仅使用胚层阶段（第 5 阶段）的胚胎12.对于 PGC 收集，我们通常在 20 分钟内对齐大约 40 个供体胚胎，并在早期阶段从大约 30 个胚胎中收集 PGC;不使用较老和有缺陷的胚胎。冷冻保存和解冻后，PGC应保持其形状;PGCs在保存不成功时破裂。宿主胚胎也应处于第 5 阶段，并具有适度的内压;胚胎在轻柔的刺激后应慢慢恢复到原来的形状。过度干燥和不充分干燥的胚胎在移植后将无法正常发育。由于 PGC 的异性移植未能在果蝇中产生配子 5,10，因此将 PGC 从多个供体胚胎移植到宿主胚胎中更有可能产生可育的成虫。为此，我们通常从每根针头大约 30 个胚胎中收集 PGC。
作为冷冻保护剂，我们尝试了不同浓度的乙二醇、二甲基亚砜和甘油以及蔗糖。我们确定含有 20% 乙二醇和 1 M 蔗糖的 EBR 是最好的13;然而，使用不同的冷冻保护剂可以改善PGC的保存22。
这种冷冻保存方法需要 PGC 处理方面的专业技能，并且需要大约 6 周的培训才能轻松收集和移植 PGC。为了评估和提高技能熟练程度，这可以分为六个训练步骤：1）在载玻片上对齐胚胎，2）控制操纵器，3）将PGC从一个胚胎移植到另一个胚胎中而不进行冷冻保存，4）将PGC从10个或更多胚胎移植到5到10个胚胎中，5）在应用CPA后移植PGC，以及6）在冻融后移植PGC。每个步骤可能需要 1 周时间。第 3 步的短期目标是孵化率为 40%，胚胎到成虫的存活率为 10%-20%，可育果蝇的频率为 20%。
PGC冷冻保存需要昂贵的仪器和高技能的人员。因此，这种方法可能不被许多实验室采用。然而，目前的PGC方法有几个重要方面。首先，PGC比胚胎小得多，并且对冷冻保护剂具有很强的渗透性。相比之下，冷冻保护剂的渗透性受到果蝇胚胎蜡质层的严重限制，这是胚胎冷冻保存中最严重的问题。事实上，以前的研究已经付出了巨大的努力，以找到一个胚胎具有高存活率和更薄蜡层的时间窗口。第二个是关于菌株之间的发育和形态变异。PGCs从早期5期胚胎（产卵后2小时30分钟-3小时20分钟）收集，而胚胎冷冻保存在16期胚胎上（产卵后14-22小时）。因此，与PGC冷冻保存相比，胚胎年龄要大得多，并且在冷冻保存的最佳时间窗口内显示出更大的菌株变异。事实上，在Asaoka等人13研究的五种菌株中，宿主产生供体衍生后代的频率没有变化，尽管宿主不是非配子的。此外，PGCs具有用于基因工程应用的潜力，例如基因组编辑14,15,16。

通过将成蝇转移到新的食品瓶中来维持果蝇菌株不可避免地导致突变的积累和表观遗传变化。开发一种长期维持果蝇菌株而不进行这种变化的替代方法势在必行，特别是对于必须维持全基因组的参考菌株。已经描述了几种冷冻保存果蝇胚胎或卵巢的成功尝试 1,2,3。不幸的是，由于可重复性低，它们仍然没有实际用途。事实上，早期胚胎在冷冻保存后的存活率较低，因为它们的卵黄含量高，这阻碍了冷冻保护剂 （CPA） 的渗透和扩散 2,3。CPA通透性也受到晚期胚胎蜡质层的严重限制。要找到一个特定菌株的时间段，在这个时间段内，胚胎具有较高的存活率和较薄的蜡层，这是困难和耗时的。最近，Zhan等4改进了胚胎透化、CPA加载和玻璃化的方法，并成功冷冻保存了多个菌株的胚胎。然而，这些方法不容易应用，因为透化后胚胎的活力往往较差。因此，仍然需要进一步改进和发展替代方法。涉及原始生殖细胞 （PGC） 冷冻保存的方法是长期维持果蝇菌株的替代方法。
PGC（也称为极细胞）移植已被用于产生生殖系嵌合体，尤其是雌性，以研究诸如合子致死突变的母体效应和生殖细胞的性别决定等过程 5,6,7,8,9,10,11,12 .PGC比胚胎小得多，并且可能对大多数冷冻保护剂具有高度渗透性。此外，菌株之间的发育和形态变异问题较少，非配子宿主可以快速恢复全基因组。我们最近开发了一种PGC冷冻保存13的新方法，该方法可以防止果蝇菌株中不可避免的遗传和表观遗传变化。在这里，我们介绍详细的协议。
这种冷冻保存方法需要 PGC 处理和仪器方面的特定专业知识。虽然对于那些不熟悉它的人来说，循序渐进的方法可能是一种有效的解决方案，但由于仪器要求，它可能不适合小型实验室。与胚胎冷冻保存方案相比，这种PGC冷冻保存方案可以更容易地适用于不同的果蝇物种和不同的昆虫物种，因为发育和形态学差异较小。PGCs还可能用于基因工程应用，如基因组编辑14,15,16。总之，这种方法可用于库存中心和其他实验室，以长时间保持苍蝇和其他昆虫菌株而不发生变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>017-00256</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>010-08725</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>038-24985</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Capillary</td>
			<td>Sutter Instrument</td>
			<td>B100-75-10-PT</td>
			<td>BOROSILICATE GLASS; O.D: 1.0mm, I.D: 0.75mm , length: 10cm, 225Pcs</td>
		</tr>
		<tr>
			<td>Capillary holder</td>
			<td>Eppendorf</td>
			<td>5196 081.005</td>
			<td>Capillary holder 4; for micromanipulation</td>
		</tr>
		<tr>
			<td>Chromic acid mixture</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>037-05415</td>
			<td>For needle washing</td>
		</tr>
		<tr>
			<td>CPA solution</td>
			<td></td>
			<td></td>
			<td>1x EBR containing 20% ethylene glycol and 1M sucrose</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>3M</td>
			<td>Scotch w-12</td>
			<td>For glue extracting</td>
		</tr>
		<tr>
			<td>Ephrussi&#8211;Beadle Ringer solution (EBR)</td>
			<td></td>
			<td></td>
			<td>130 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM Hepes at pH 6.9</td>
		</tr>
		<tr>
			<td>Ethanol (99.5)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>057-00451</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>054-00983</td>
			<td>For CPA solution</td>
		</tr>
		<tr>
			<td>Falcon 50 mm x 9 mm bacteriological petri dish</td>
			<td>Corning Inc.</td>
			<td>351006</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Vigor</td>
			<td>Type5 Titan</td>
			<td>For embryo handling</td>
		</tr>
		<tr>
			<td>Grape juice</td>
			<td>Asahi Soft Drinks Co., LTD.</td>
			<td>Welch&#39;s Grape 100</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Grape juice agar plate</td>
			<td></td>
			<td></td>
			<td>50% grape juice, 2% agar, 1% ethanol, 1% acetic acid</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>084-08105</td>
			<td>For glue extracting</td>
		</tr>
		<tr>
			<td>Humidifier</td>
			<td>APIX INTERNATIONAL CO., LTD.</td>
			<td>FSWD2201-WH</td>
			<td>For embryo preparation</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica Microsystems GmbH</td>
			<td>Leica&#160;DM IL LED</td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Luer-lock glass syringe</td>
			<td>Tokyo Garasu Kikai Co., Ltd.</td>
			<td>0550 14 71 08</td>
			<td>Coat a plunger with silicon oil (FL-100-450CS);for micromanipulation</td>
		</tr>
		<tr>
			<td>Mechanical micromanipulator</td>
			<td>Leica Microsystems GmbH</td>
			<td></td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Micro slide glass</td>
			<td>Matsunami Glass Ind., Ltd.</td>
			<td>S-2441</td>
			<td>For embryo aligning</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE Group</td>
			<td>Custom order</td>
			<td>EG-401-S combined EG-401 and MF2 (with ocular lens MF2-LE15 ); for needle preparation</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica Microsystems GmbH</td>
			<td>Leica MC170 HD</td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Merck KGaA</td>
			<td>Eppendorf TransferTip (ES)</td>
			<td>For cryopreservation</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>28514-75</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>NARISHIGE Group</td>
			<td>PN-31</td>
			<td>For needle preparation; the heater level is set to 85.0-98.4, the magnet main level to 57.8, and the magnet sub level to 45.0.</td>
		</tr>
		<tr>
			<td>PVC adhesive tape for electric insulation</td>
			<td>Nitto Denko Corporation</td>
			<td>&#160;J2515</td>
			<td>For embryo-pool frame</td>
		</tr>
		<tr>
			<td>Silicon oil</td>
			<td>Shin-Etsu Chemical, Co, Ltd.</td>
			<td>FL-100-450CS</td>
			<td>For embryo handling</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>31320-05</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>197-02206</td>
			<td>Undiluted and freshly prepared; for embryo breaching</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>30404-45</td>
			<td>For CPA solution</td>
		</tr>
	</tbody>
</table>

primordial germ cell cryopreservation and revival of drosophila strains

果蝇 菌株必须通过频繁地将成年果蝇转移到新的小瓶中来维持。这带来了突变恶化和表型变化的危险。因此，开发一种无需此类更改即可长期保存的替代方法势在必行。尽管以前有过成功的尝试，但由于可重复性低， 果蝇 胚胎的冷冻保存仍然没有实际用途。在这里，我们描述了一种通过将冷冻保存的PGC移植到非配子 果蝇黑腹 果蝇（D. melanogaster）宿主胚胎中来冷冻保存和菌株复兴的方案。PGC对冷冻保护剂（CPA）具有高度渗透性，菌株之间的发育和形态变异比胚胎冷冻保存中的问题要小。在这种方法中，从大约30个供体胚胎中收集PGC，在CPA处理后加载到针中，然后在液氮中冷冻保存。为了产生供体来源的配子，将针头中冷冻保存的PGC解冻，然后沉积到大约15个非配子宿主胚胎中。使用该协议实现了至少15%的可育果蝇的频率，并且每对可育夫妇的后代数量总是足以恢复原始菌株（平均后代数量为77.2±7.1），表明冷冻保存的PGC具有成为生殖干细胞的能力。每针平均可育果蝇数量为1.1±0.2只，26根针中有9只产生两个或两个以上的可育后代。结果发现，11根针足以产生6个或更多的后代，其中可能至少包括一名雌性和一名雄性。非配属宿主可以通过简单地将新出现的雌性和雄性苍蝇杂交来快速恢复菌株。此外，PGC具有用于基因工程应用的潜力，例如基因组编辑。

The maintenance of Drosophila strains by the transfer of adult flies to new food vials inevitably results in the accumulation of mutations and epigenetic changes over time. Development of an alternative method for long-term maintenance of Drosophila strains without such changes is imperative, especially for reference strains in which the whole genome has to be maintained. Several successful attempts to cryopreserve Drosophila embryos or ovaries have been described1,2,3. Unfortunately, they are still not of practical use because of low reproducibility. Indeed, early-stage embryos have a low survival rate after cryopreservation because of their high yolk content, which impedes cryoprotective agent (CPA) permeation and diffusion2,3. CPA permeability is also severely limited by the waxy layers of late-stage embryos. It is difficult and time-consuming to find a strain-specific time period in which embryos have a high survival rate and a thinner wax layer. Recently, Zhan et al.4 improved methods for embryo permeabilization, CPA loading, and vitrification and successfully cryopreserved embryos of multiple strains. However, the methods are not easy to apply because the viability of embryos after permeabilization tends to be poor. Therefore, further improvement and development of alternative approaches are still needed. Methods involving the cryopreservation of primordial germ cells (PGCs) are an alternative approach for the long-term maintenance of Drosophila strains.
PGC (also called pole cell) transplantation has been used to generate germline chimeras, especially females, to study processes such as maternal effects of zygotic lethal mutations and sex determination of germ cells5,6,7,8,9,10,11,12. PGCs are much smaller than embryos and are likely to be highly permeable to most cryoprotectants. Furthermore, developmental and morphological variation among strains is less problematic, and an agametic host enables quick restoration of whole genomes. We recently developed a new method of PGC cryopreservation13, which prevents the otherwise inevitable genetic and epigenetic changes in Drosophila strains. Here, we present the detailed protocol.
This cryopreservation method requires specific expertise in PGC handling and instrumentation. While a step-by-step approach may be an efficient solution for those who are unfamiliar with it, it may be unsuitable for small laboratories due to instrumentation requirements. This PGC cryopreservation protocol can be more easily adapted for use with different Drosophila species and different insect species than embryo cryopreservation protocols because of smaller developmental and morphological differences. PGCs can also potentially be used in genetic engineering applications, such as genome editing14,15,16. In summary, this method can be used in stock centers and other laboratories to maintain fly and other insect strains for prolonged periods of time without changes.

作者聚焦：PGC 冷冻保存 – 一种长期保存果蝇菌株的替代方法 

果蝇菌株的原始生殖细胞冷冻保存和复兴

Drosophila strain must be maintained by the frequent transfer of adult flies to new vials. This creates a danger of mutational deterioration and genetic changes. We aim to develop an alternative method for the long-term preservation of Drosophila strains without such changes.Compare with in vivo editing, genome editing of primordial germ cells, or PGCs, could increase knock-in inefficiency and make more complicated genomic changes. Thus, a wide application of PGC manipulation facilitates genetic engineering in many fields. We now focus on xenotransplantation.By using Drosophila melanogaster as a surrogate host, we have succeeded in reviving stock of cell species in the melanogaster species subgroup. By addressing xenotransplantation barriers and finding possible solutions, we would like to explore the potential application of PGC transplantation and cryopreservation.

Asaoka 等人 13 报道了冷冻保存的 PGC 移植的效率，并在 表 2 中给出了在液氮中冷冻保存 1 天或更长时间的 PGC 移植。孵化率为168/208个移植胚胎（80.8%），胚胎到成体的存活率为87/208个（41.8%）。可育果蝇的发生率为28/87（32.2%）。冷冻保存 8 至 30 天的 PGC 和冷冻保存 31-150 天的 PGC 之间，该频率没有差异（20/57 vs. 8/30，G' = 0.63，p >0.1，d.f. = 1）。每对夫妇的平均后代数为77.2±7.1（n = 18,28-122），表明冷冻保存的PGCs具有成为生殖干细胞的能力。在26针中，10针没有产生可育后代，7针产生1个可育后代，7针产生2个可育后代，2针产生3或4个可育后代。每针平均可育苍蝇数量为1.1±0.2只。根据这些数据，有 95% 的置信度，11 根针足以产生 6 个或更多的后代，其中可能至少包括一个雌性和一个雄性。
在上述实验中，我们使用PGCs（nanos>ovo-A，OvoA_OE胚胎）中表达ovo-A mRNA的胚胎作为非配子宿主。在移植的纳米>卵-A夫妇产生的669只F1雌性和720只F1雄性中，没有来自宿主PGC的逃逸者。几个oskar（osk）突变体也是温度敏感的非配子20,21。由于不再有具有高纯合子活力和非配子表型的osk突变体，我们通过CRISPR / Cas9辅助的基因组编辑重新创建了osk[8]错义突变体20。这些苍蝇在25°C时完全是非配子的（230只雌性中的0只逃逸者和192只雄性），但在23°C时出现了一些逃逸者（248只雌性中的1只和290只雄性中的1只）。因此，nanos>ovo-A被推荐为非配子宿主胚胎。UASp-ovo-A 和 nanos-Gal4 库存13 都将很快从京都果蝇库存中心获得。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65985/65985fig01v2.jpg"> 图 1：所需设备。 （A） 用于收集和移植细胞的显微操纵系统。i）倒置显微镜，ii）机械显微操纵器，iii）注射器，iv）毛细管支架，v）三通旋塞阀，vi）加湿器，vii）体视显微镜。（B） 注射器。（C） 三通旋塞阀和硅胶管连接注射器和毛细管支架。（D） 将针头和毛细管支架连接到显微操纵器上。（E） 带有胚胎收集板的胚胎收集杯（直径6厘米，高7.7厘米）。（F） 不锈钢网过滤器。（G） 用作带有载玻片的湿腔室的容器。为保持湿度，请将湿纸放在底部并盖上盖子。（H） 带有用于冷冻保存的针头的针架。（一）冷冻保存的储存架和带针头的盒子。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig01largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65985/65985fig02v2.jpg"> 图 2：PGC 收集载玻片和冷冻保存针。 （A） 涂有胶水的原始生殖细胞 （PGC） 收集载玻片。去角质胚胎排列成两排，并使其前部向右（要操作的一侧）和腹侧朝上。固定胚池框架，沉积两滴冷冻保护剂 （CPA） 溶液，并在池中填充硅油。（B） 针头应含有尽可能少的蛋黄和其他污染物。当PGC在液氮中冷冻保存时，PGC夹在两层硅油之间。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig02largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65985/65985fig03.jpg"> 图 3：制作针。 三步针尖抛光方法，使针具有合适的孔径和锋利的针头。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65985/65985fig04v2.jpg"> 图4：胚胎收集方案。 经过两次预收集后，我们通常每天收集三到四次。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig04largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65985/65985fig05.jpg"> 图5：宿主胚胎比对。 在载玻片上对准宿主胚胎。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65985/65985fig06.jpg"> 图 6：PGC 冷冻保存方法概述。 概述进行原始生殖细胞 （PGC） 冷冻保存所遵循的所有步骤。 <a href="https://www.jove.com/files/ftp_upload/65985/65985fig06large.jpg" target="_blank">请点击这里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></td>
			<td colspan="3">室内湿度</td>
		</tr><tr><td></td>
			<td>< 30%</td>
			<td>~ 30%</td>
			<td>> 30%</td>
		</tr><tr><td>对齐宿主胚胎 （~20 分钟）</td>
			<td>使用加湿器 2 - 10 分钟</td>
			<td>间歇性使用加湿器 1 分钟</td>
			<td>不要使用加湿器</td>
		</tr><tr><td>解冻供体PGC</td>
			<td>不適用</td>
			<td>不適用</td>
			<td>不適用</td>
		</tr><tr><td>风干PGC</td>
			<td>省略此步骤</td>
			<td>省略此步骤</td>
			<td>5 分钟</td>
		</tr><tr><td>涂抹硅油</td>
			<td>不適用</td>
			<td>不適用</td>
			<td>不適用</td>
		</tr><tr><td>移植PGC</td>
			<td>不適用</td>
			<td>不適用</td>
			<td>不適用</td>
		</tr><tr><td colspan="4">所有这些步骤都应在 50 分钟内完成。</td>
		</tr></tbody></table>表1：胚胎比对和PGC解冻期间胚胎的干燥。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>供体菌株</td>
			<td>冷冻保存期</td>
			<td>移植胚胎数量（A）</td>
			<td>孵化幼虫数量 （B） （孵化率，B/A）</td>
			<td>封闭式成人人数 （C） （卵子到成虫的生存能力，C/A）</td>
			<td>可育成虫数量 （D） （可育苍蝇的频率，D/C）</td>
		</tr><tr><td>M17型</td>
			<td>8 - 30 天</td>
			<td>134</td>
			<td>108 (80.6%)</td>
			<td>57 (42.5%)</td>
			<td>20 (35.1%)</td>
		</tr><tr><td>M17型</td>
			<td>31 - 150 天</td>
			<td>74</td>
			<td>60 (81.1%)</td>
			<td>30 (40.5%)</td>
			<td>8 (26.7%)</td>
		</tr><tr><td colspan="6">M17： yw;TM6B， P{Dfd-GMR-nvYFP}4， Sb[1] Tb[1] ca[1]/ Pri[1]</td>
		</tr></tbody></table>表2：冷冻保存PGC移植的效率。 此表修改自13.所有数据均来自非游戏主机。

Watch this Scientific Journal Video about PGC Cryopreservation – An Alternative Approach Towards Long-Term Preservation of Drosophila Strains at JoVE.com

果 蝇 菌株的长期保存方法作为成年苍蝇频繁转移到新鲜食品瓶的替代方案是非常可取的。该方案描述了 果蝇 原始生殖细胞的冷冻保存和菌株通过移植到非配子宿主胚胎的复兴。

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

果蝇菌株必须通过频繁地将成年苍蝇转移到新的小瓶中来维持。这造成了突变恶化和遗传变化的危险。我们的目标是开发一种替代方法，以便在不进行此类更改的情况下长期保存果蝇菌株。与体内编辑相比，原始生殖细胞（PGC）的基因组编辑可能会增加敲入效率并产生更复杂的基因组变化。因此，PGC操作的广泛应用促进了基因工程在许多领域的应用。我们现在专注于异种移植。通过使用黑腹果蝇作为替代宿主，我们成功地恢复了黑腹果蝇物种亚群中的细胞物种种群。通过解决异种移植障碍并寻找可能的解决方案，我们希望探索PGC移植和冷冻保存的潜在应用。

primordial-germ-cell-cryopreservation-revival-drosophila

Research

JoVE Journal

Genetics

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15&#160;agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 &#177; 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 &#177; 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

科研

遗传学

Collection of Primordial Germ Cells from Drosophila Donor Embryos

Collection of Primordial Germ Cells from Drosophila Donor Embryos  

Proceed to collect the primordial germ cells or PGCs from the dross filia embryos. After assembling the micro manipulator system. While working under a stereo microscope, use forceps to transfer the embryos.Align the coated embryos in two rows on a PGC collection glass slide along two reference lines. Orient the embryos with their anterior to the right and ventral side up. Gently move the transplantation needle tip to the anterior end of an embryo.Penetrate the embryo by moving the microscope stage and retract the needle slightly. When the tip reaches the posterior end. Discharge any yoke in the needle inside the somatic cell layer.Move the needle tip to the posterior pole and gently load the PGCs into the needle without taking much time. Pull the needle out of the embryo quickly. Discharge yolk and other contaminants from the needle while keeping the PGCs.Load clean silicone oil from the pool into the needle. Before collecting PGCs from a new embryo, deposit as much silicone oil as possible inside the somatic cell layer while keeping the loaded PGCs in the needle. After collecting PGCs from all embryos in a row, deposit the PGCs onto the surface of an embryo and unload any yoke or other contaminants onto another neighboring embryo.In a similar manner, collect and combine PGCs from the embryos in the second row.

从 果蝇 供体胚胎中收集原始生殖细胞

Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor Embryos

Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor Embryos  

Proceed for the cryo-preservation of the primordial germ cells or PGCs. After collecting and depositing the PGCs from the donor embryos on the surface of an embryo. Wash the transplantation needle with a cryo protective agent or CPA and load fresh CPA into the needle.Add the loaded CPA to the PGCs deposited on the embryo while ensuring the volume of CPA is equivalent to that of the PGCs. Remove excess CPA from the cluster of PGCs one to two seconds after CPA addition. Next, empty the needle and load it with silicone oil for five seconds or longer.Load all the collected PGCs into the needle, followed by loading silicone oil again for five seconds or longer. Ensure the PGCs are sandwiched between two layers of silicone oil. Detach the needle from the micro manipulator using soft tissue paper, blot the oil off the needle surface without touching the needle tip with the tissue.Attach the needle to a needle holder and using vinyl tape, lock it in position at the base. Flash freeze the holder with the needle pointing downwards by submerging it in liquid nitrogen. Do not release the holder until the liquid stops fizzing out of the rack.Store the holder in a liquid nitrogen storage tank in the liquid phase area.

从 果蝇 供体胚胎中收集的原始生殖细胞的冷冻保存

Thawing and Transplantation of Cryopreserved Primordial Germ Cells for Drosophila Strain Revival

Thawing and Transplantation of Cryopreserved Primordial Germ Cells for Drosophila Strain Revival  

To begin, align the stage five egg mimetic Drosophila host embryos on a transplantation glass slide. While orienting their posterior to the right and ventral to the top, line up approximately 30 embryos in two rows in 20 minutes. Thaw the cryopreserved PGCs in the needle by submerging the holder with the needle pointing downward into EBR solution at room temperature.Keep it submerged for 10 seconds. Place the transplantation glass slide on the microscope stage. Attach the freeze thawed needle to the capillary holder and bring the first embryo into the left row and the needle tip into the same focal plane.Using a 20x objective lens, position the needle tip gently at the surface of the posterior end of the embryo. Prod the outside of each embryo gently, and confirm that they slowly return to their original shape. Penetrate an embryo from the posterior pole using the needle.Gently deposit approximately 10 to 20 PGCs inside the posterior pole, specifically between the vitelline membrane and the somatic cell layer, and retract the needle from the embryo before repeating the same for subsequent embryos. Finally, to revive the strain, cross newly emerged females and males.