我们感谢与 Ju 实验室和中实验室的成员进行了深思熟虑的讨论, 以及莱特州立大学的财政支持。

注: 此处描述的协议用于筛选十96井板中包含的库质粒, 但可以相应地放大或缩小。需要重复该协议以完成整个图书馆的筛选。通常情况下, 每次对10块的图书馆基因进行筛查, 可以得到1人的舒适处理。
1. 96 井酵母转化剂的制备
注意: 此步骤按照前面描述的7、10进行。
<ol><li>整除5µL (约 50–100) 从阵列质粒库中的质粒 DNA 到圆底96井板的每一井。 注: 这样一个图书馆的例子是酵母基因过度表达图书馆10。</li>
	<li>接种150毫升的酵母蛋白胨葡萄糖 (YPD) 培养基, 在500毫升的烧瓶中有一个菌落 (2–3毫米直径) 的单倍体酵母菌株 W303α。一夜之间孵化他们在30°c 与震动 (200 转每分钟)。</li>
	<li>第二天早晨, 测量隔夜文化的 od600并且稀释酵母文化到 OD600 = 0.1 在 (至多) 2 L YPD。孵化它在30°c 与震动 (200 转每分钟) 为 ~ 5 h 直到文化到达 OD600 = 0.4–0.6。</li>
</ol>2. 酵母转化
<ol><li>通过填充8无菌250毫升离心机瓶和离心他们在室温下 3000 x g为10分钟来收获酵母培养. 在不破坏球团的情况下倒入上清液。 注: 在室温下执行所有离心步骤。</li>
	<li>用无菌蒸馏的 H2O 洗净酵母。为此, 增加100毫升不育 H2O 到每个离心机瓶和漩涡它并用重悬细胞颗粒。将洗涤过的细胞组合成2瓶, 并将其离心在 3000 x g处以5分钟。</li>
	<li>在每瓶100毫升的 0.1M LiOAc/1XTE (100 毫米 LiOAc; 10 毫米三, pH 8.0; 1 毫米 EDTA) 中清洗每个瓶子中的细胞, 并将其离心在 3000 x g上, 以5分钟为单位倒入上清。</li>
	<li>当离心, 煮沸5毫升鲑鱼精子 DNA (10 毫克/毫升) 在100°c 使用一个块加热器3分钟, 然后冷却它在冰上。</li>
	<li>并用重悬细胞颗粒在每瓶25毫升 0.1M LiOAc/1XTE, 结合悬浮细胞和转移到一个150毫升烧瓶。添加5毫升的预冷鲑鱼精子 DNA 和孵化它在30°c 与震动 (225 rpm) 30 分钟。</li>
	<li>将细胞混合物倒入无菌的一次性试剂库中, 使用多通道吸管, 将细胞混合物的35µL 转移到含有库质粒 DNA 的圆底96井板的每个井中。涡流96井板使用一个板块 vortexer 1 分钟在 1000 rpm。在30摄氏度的情况下孵育30分钟, 不摇晃。 注意: 不要堆叠板, 这样热量就能更有效地转移。我们发现, 涡流的96井板在 1000 rpm 不会导致液体溢出的油井, 但一个安全的涡流速度应该在执行步骤之前测试。</li>
	<li>在烧瓶中, 准备200毫升的转换缓冲器, 其中包含最终浓度为 40% PEG3350, 10% 亚砜和 0.1M LiOAc。在使用前立即准备转换缓冲器, 并通过摇动彻底混合。</li>
	<li>从30°c 孵化器中删除96井板, 并将其混合三十年代在 1000 rpm 使用板 vortexer。将转换缓冲器的125µL 添加到每个井中, 然后在1000转每分钟旋转1分钟的平板。</li>
	<li>孵化的板块在30°c 30 分钟, 然后热休克酵母通过放置在42°c 孵化器的15分钟。 注意: 不要堆叠板。</li>
	<li>在 3000 x g上离心板5分钟。通过在垃圾桶上反转板块, 并强行从盘子中倾倒缓冲物, 从油井中取出转换缓冲器。迅速将倒置的盘子涂抹在干净的纸巾上, 以除去盘子顶部的液体。</li>
	<li>将细胞添加到200µL 的最小脱落介质中, 相应于库质粒的可选标记。 注: 我们使用了一个综合市建局-媒介。</li>
	<li>将板材离心5分钟, 在 3000 x g处, 在垃圾桶上取出上清液, 如步骤2.10 所明。</li>
	<li>添加160µL 的最小的市建局-培养基含2% 葡萄糖。涡流板1分钟在 1000 rpm 和孵化他们在30°c 48 小时, 而不动摇。48小时后, 请注意, 在每个井的底部可见一个转化后的酵母颗粒。</li>
	<li>使用液体分配器, 添加100µL 的市建局-媒体含有葡萄糖的每一个新的一套96井板。</li>
	<li>涡流的板块包含转化酵母 (从步骤 2.13) 在1000转每分钟三十年代. 使用无菌塑料96针复制器, 将引脚放入含有转化酵母的井中, 然后将其接种到新板的相应井中。充满了媒体。孵化新的板材在30°c 为24小时。 注: 如果使用10板, 介质也可以用多通道吸管手动配药。</li>
	<li>24小时后, 每个井的底部都应可见一个酵母颗粒, 其中含有成功转化的酵母。为了保存这些酵母菌株作为甘油股票, 添加50µL 50% 甘油每一个井, 漩涡的板块为三十年代在 1000 rpm, 密封的盘子与密封胶带和冻结他们在-80 °c。 注意: 以上步骤只需要执行一次。不同酵母模型的未来筛选对同一图书馆一次性试剂库可以从甘油库存开始, 并立即从以下步骤进行。</li>
</ol>3. 含有图书馆基因的细胞与查询酵母的交配
<ol><li>要从甘油库存中恢复库株, 从-80 °c 冰箱取出96井板, 取出密封胶带, 让酵母在室温下解冻约30分钟。</li>
	<li>一旦酵母解冻, 使用无菌塑料96针复制器接种甘油股票到160µL 新鲜的市建局-媒体包含2% 葡萄糖在96井板和孵化他们在30°c 为 24 h. 在使用库菌株后立即, 用密封胶带封住盘子, 并将其退回到-80 摄氏度冰柜。</li>
	<li>在同一天图书馆菌株 (W303α) 解冻, 接种查询酵母菌株 (这里, FUS 在 W303a) 到50毫升 YPD 和增长它隔夜在30°c 与震动在250转每分钟。</li>
	<li>第二天早上, 将查询酵母菌倒入一个无菌的一次性试剂库, 用多通道吸管将查询菌株的160µL 整除到96井板的每一个井中。</li>
	<li>使用液体分配器, 将160µL 的 YPD 介质放入十96井板的每一个井中。使用这些板块以后交配的查询酵母菌株与图书馆酵母。</li>
	<li>简要地将含有查询应变的96井板涡旋, 然后使用无菌96针复制器将查询应变转移到 YPD 板上。</li>
	<li>简要地涡旋的图书馆应变板, 并为每个图书馆的应变板, 使用新的无菌96针复制器转移到 YPD 板, 已接种的查询应变。 注: 甘油细胞在多次解冻冷冻循环后失去活力。尽快将图书馆的应变恢复到长期储存 (-80 °c 冷冻库), 使图书馆保持良好的质量, 并准备好将来使用。适当维持, 甘油的库存可以冻结和解冻至少10倍。我们还强烈建议保存甘油库存的工作副本和备份副本。当工作库存不工作时, 立即从备份副本中制作新的工作副本。</li>
	<li>孵化的 YPD 板在30°c 24 小时, 请注意, 酵母颗粒将在每井底部可见24小时后。</li>
	<li>填充96井板, 其最小的脱落介质含有2% 糖, 对应于查询菌株中质粒的可选择标记以及库质粒 (例如, 市建局-他的-)。</li>
	<li>使用无菌的96针复制器将酵母从 YPD 交配培养基转移到选择性培养基上。孵育96井板在30°c 为 48 h;只有有交配和形成双细胞的酵母细胞, 因此, 含有的查询质粒和库质粒将能够生长在这个媒介。2天后, 观察在油井底部可见颗粒。</li>
</ol>4. 斑点测定法
<ol><li>
在含糖的选择性介质中, 经过48小时的生长, 在琼脂盘上发现酵母。
	<ol>
<li>准备2套市建局-他的辍学板含有2% 琼脂, 使用透明聚苯乙烯板, 其中含有2% 半乳糖, 另一个含有2% 葡萄糖。</li>
		<li>涡96井板1分钟在1000转, 然后发现酵母到市建局-他的辍学板包含2% 半乳糖和2% 琼脂 (FUS 和图书馆基因被诱导) 和对市建局-他的辍学板包含2% 葡萄糖和2% 琼脂 (FUS 和图书馆基因压制) 使用机器人的识别机, 其中每个井的文化被发现到四份的琼脂板块 (即, 1 井的文化被发现到4斑点的琼脂板)。</li>
		<li>发现后, 让琼脂板块干燥, 然后把它们倒在30摄氏度孵化器。每24小时拍摄琼脂板块, 以记录一个更快/更多的酵母生长, 其中的毒性, 以查询菌株被拯救或记录一个较慢/少的增长, 其中对查询菌株的毒性加剧。将盘子孵化4天。 注: 每个井中的文化可以在1到1的琼脂板上发现, 如以前的基于变换的方法10所示。然而, 这里的1到4的发现 (可以方便地使用机器人识别机设置) 显著增加了检测的鲁棒性, 减少误报的数量。只有当所有4个来自同一井的菌落表现出相似的表型时, 才会考虑正面撞击。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个在酵母中执行质粒过度表达筛的协议, 将质粒库引入到酵母模型中。利用这种方法, 可以用同质粒库转化的同一株酵母, 筛选出多种神经退行性疾病蛋白毒性酵母模型。转化过程只需要进行一次, 然后用高效的酵母交配将质粒库引入到查询菌株中。该协议确实依赖于使用机器人设备来分配媒体和斑点酵母培养到琼脂板块。虽然可以在不使用机器人设备的情况下执行该协议, 但这将会耗费更多的时间。该方法成功地用于筛选能改变 FUS 毒性的基因。
我们观察到, FUS 在双倍酵母的背景下毒性较小。这很可能是由于基因复制数量和不同的增长速度的双倍酵母。除非正在研究的表型为交配型或倍性依赖性, 否则毒性的生长表型在单倍体和双酵母中是一致的。因此, 基于交配的方法有望在多种生长表型的酵母模型中得到广泛的应用。然而, 应该验证酵母模型的表型, 以确保在进行这种筛选方法之前, 它仍然存在于双倍的背景中。该方法可用于酵母中多种不同表型的研究, 不限于神经退行性蛋白毒性的研究。此外, 任何含有酵母表达载体的质粒库都可以使用。
在执行屏幕后, 有许多验证化验, 将有助于确保确定的命中是特定的酵母模型被筛选。应在酵母中进行毒性增强剂的试验, 而不同时表达感兴趣的疾病蛋白。促进剂引起的毒性无关的疾病蛋白的利益, 应消除进一步研究。重要的是要考虑毒性抑制因素是否影响了疾病蛋白的表达, 影响 Gal1 启动子。任何影响 Gal1 启动子表达的抑制因素都应被排除在进一步研究之外。
含有库质粒的酵母菌被永久储存在甘油库存中, 在需要的时候可以迅速恢复, 以便以交配为基础的方法可以很容易地应用到其他酵母模型中, 同样的库基因需要筛选。当采用相同类型的筛选去除假阳性, 或需要研究多种不同的酵母模型时, 基于交配的方法的效率变得明显。我们已经成功地用这种方法筛选了 TDP-43 的酵母模型, 另一个与 ALS 相关的蛋白质。

酵母酵母菌在 1基础科学研究中得到广泛应用, 以了解与人类疾病直接相关的细胞过程。此外, 它被用作研究人类疾病相关蛋白的模型有机体, 例如与最常见的神经退行性疾病相关的蛋白质, 包括阿尔茨海默病、帕金森病、亨廷顿氏病和肌萎缩症。侧索硬化症 (ALS)2。酵母模型的优点是可以轻松地使用一个基因组范围的屏幕来识别与疾病相关蛋白的毒性相关的细胞通路, 从而对其毒性机制进行深入的研究。一个这样的屏幕被称为一个过度表达的图书馆屏幕, 其中5500酵母基因的排列库被转化为酵母模型, 以确定哪些基因可以修改毒性时抗原。这种筛选方法已成功地应用于多种神经退行性疾病相关蛋白的酵母模型中, 包括亨廷顿氏病的杭丁顿蛋白3、4、5 的α synuclein., Aβ阿尔茨海默氏病6, FUS 和 TDP-43 为 ALS7,8,9。虽然它通常是在高通量的方式10, 最劳动密集型的步骤的屏幕是单独转换5500酵母基因从阵列库。这一步必须在每次重复筛选时执行, 并且每当需要研究新建立的酵母模型时。找到一个更有效的方法来完成这项任务是很重要的。
酵母细胞可以稳定地存在于单倍体和双体形态中。单倍体细胞有两种相反的交配类型, 交配型 a 和α. 每种交配类型的单倍体细胞产生和分泌它们自己特定的交配信息素, 只有相反的交配型细胞反应。这允许在 a 和α细胞之间交配产生稳定的双倍细胞, a/α。这个过程是自发和高效率的11。我们可以利用这种独特的酵母生命周期来介绍质粒库。更具体地说, 阵列质粒库中的每个基因都将转化为一个交配型的单倍体细胞,即α细胞。这些含有库基因的细胞将会以96井格式排列的甘油储存。对于每个需要筛选的酵母模型, 含有该库基因的酵母细胞可以从甘油类中解冻, 通过与交配类型的感兴趣的酵母模型交配来进行筛选,即交配型 a。这种使用交配把两个基因放到酵母中的想法并不是新的。它已成功地应用于高通量酵母双杂交筛选中, 其中一个交配类型的诱饵构造 (即Gal4-DNA-binding 域融合) 通过与一个阵列库的猎物构造交配而聚集在一起。12. 然而, 这一战略从来没有被应用于过度表达的图书馆放映中, 因为它一直采用传统的转换方法。
我们的实验室以前建立了一个酵母模型的 ALS 相关蛋白 FUS7。通过使用转换方法进行过表达库筛选, 我们发现了五种酵母基因 (ECM32、NAM8、SBP1、SKO1和VHR1), FUS 时抢救抗原的毒性。这些发现被独立地证实了以相似的研究由另一组8。hUPF1, 人类同源的ECM32, 后来被证明抑制毒性的主要神经细胞13和在动物模型 ALS14以及。利用这五种基因作为原理证明, 我们证明, 在 FUS 酵母模型中, 通过交配, 所有的五种基因都类似地拯救了 FUS 毒性。由于含有库基因的酵母细胞可以永久储存在甘油库存中, 需要时恢复, 这种基于交配的方法将在每次需要筛选库时消除耗时的转换步骤。由于交配效率高, 不涉及质粒的转化, 这一策略也大大降低了大型质粒库的纯化和转化所带来的成本。我们将成功地将此方法应用于 FUS 酵母模型的图书馆筛选。
图 1简要介绍了基于交配的筛查程序。最初, 阵列质粒库被转化成一个单倍体酵母菌株的交配型α使用高通量酵母转化协议, 其中每个井的96井板含有酵母转化为特定的图书馆质粒。这种转化酵母的收集保存为甘油的股票, 可以解冻和恢复使用后。酵母模型的兴趣, 在这种情况下, FUS 毒性, 必须产生的单倍体酵母菌株与相反的交配类型 (交配型 a)。利用无菌96针复制器的高通量方式, 将含有质粒库的 FUS 菌株和酵母菌转移到含有丰富培养基的96井板上, 并允许交配。交配后, 每一个井的交配培养的小体积转移到96井板含有合成的辍学媒体, 其中只有双酵母含有 FUS 和图书馆的基因可以增长。然后利用机器人识别机将各井的酵母培养物转移到琼脂板上, FUS 和库基因的表达。 此外, 酵母培养被发现控制琼脂板块在那里 FUS 和图书馆基因没有表达。继琼脂板块生长后, 会发现拯救或加剧 FUS 毒性的基因。

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mating-based overexpression library screening in yeast

发芽酵母作为研究与人类疾病相关的蛋白质的模型被广泛应用。全基因组基因筛选是酵母研究中常用的有力工具。一些神经退行性疾病相关蛋白在酵母中的表达导致细胞毒性和聚集形成, 综述发现这些疾病的患者。在这里, 我们描述了一种筛选肌萎缩侧索硬化相关蛋白 FUS 酵母模型的方法, 以修饰其毒性。这个新的筛选平台不是使用转换, 而是依赖于酵母的交配来引入酵母模型中的粒度排列库。这种交配方法有两个明显的优点: 一是高效;二是将质粒预转换阵列库长期保存为甘油库存, 并可快速应用于其它筛网, 而不需要将劳动密集型的步骤转化为酵母模型。我们展示了这个方法如何能够成功地用于筛选基因, 改变 FUS 的毒性。

ALS 相关蛋白 FUS, RNA/DNA 结合蛋白, 以前研究的单倍体酵母7,8。遗传筛选采用基于转化的方法发现了几种抑制 FUS 毒性的酵母基因。其中一个酵母基因的人同源, 后来被证明是有效的抑制毒性的主要神经元细胞和大鼠模型的 ALS13。在这里, 我们使用相同的酵母模型表明, 过度表达的图书馆筛选可以通过交配有效地进行转换。
FUS 对单倍体和双酵母细胞都有毒性 在单倍体细胞的背景下, 对 FUS 的前酵母模型和随后的过度表达文库进行了筛选。以交配为基础的方法工作, FUS 毒性需要证明在双酵母。为此, 我们交配的 FUS 酵母模型在 w303a (交配型 a) 与 w303α转化为空向量 (交配型α)。如图 2所示, 虽然不像单倍体酵母那么强, 但 FUS 毒性在双倍酵母中明显可见。
在双倍酵母中发现的抑制基因 作为基于交配方法的原理的证明, 我们测试了以前从基于变换的方法中发现的五种基因。采用交配方法将五种基因分别引入 FUS 的单倍体酵母模型中 (W303a), 并对其在随后的双倍酵母中 FUS 的毒性进行了试验 (W303a/α)。如图 3所示, 所有五种基因都能挽救 FUS 在酵母中的毒性, 表明交配方法是有效的。
940种基因的先导筛查 (排列在十96井板上) 根据上述协议, 我们将基于交配的方法应用于940基因的无表达库筛选。图 4显示了一个有代表性的板块的图片。如图右侧所示 (FUS 和库基因表达), FUS 对酵母菌有毒性。由绿方所示的库基因救出 FUS 毒性, 而红方则增强毒性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57978/57978fig1.jpg"> 图 1: 用交配法筛选蛋白质毒性酵母模型的示意图.一种质粒库 (在半乳糖存在下高度诱导的 GAL1 启动子的控制下) 通过高通量转换协议转化为单倍体酵母菌株 (MATα)。这收集的酵母, 转化为图书馆质粒, 被储存作为甘油的股票在-80 °c, 并恢复时, 需要配合的单倍体酵母模型的蛋白质毒性 (在我们的情况下, 一个副本 FUS 集成在 HIS3 轨迹, GAL1 启动子, 马马)。选择含有库质粒和毒性蛋白 (FUS) 的双倍酵母, 发现葡萄糖 (FUS 和文库基因 ' 脱落 ') 和半乳糖 (FUS 和库基因 ' on ') 琼脂板块。酵母的生长被跟随辨认基因抢救或恶化 FUS 的毒性。绿色广场表明一个例子的抑制基因, 抢救 FUS 毒性, 红方表明一个例子的增强基因, 加剧了 FUS 毒性, 当抗原。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig1large.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/57978/57978fig2.jpg"> 图 2: FUS 对单倍体和双酵母细胞都有毒性.用 pRS303Gal1-FUS 转化成的单倍体酵母 (w303), 或者用空质粒转化, 或与相同的空质粒转化为交配型 (w303 MATα) 酵母, 产生双倍酵母菌株。这些酵母菌株连同控制菌株, 然后5x 连续稀释 (从左到右), 并发现到市建局-他的葡萄糖培养基 (左, FUS 表达压抑) 和市建局-他半乳糖培养基 (右, FUS 表达诱导)。这张照片是在2天的增长30摄氏度后拍摄的。观察到单倍体和倍性控制菌株的生长几乎相同, 只显示单倍体控制菌株。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig2large.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/57978/57978fig3.jpg"> 图 3: 五酵母基因 (ECM32、NAM8、SBP1、SKO1、 VHR1) 通过交配方法抢救 FUS 毒性.含有先前鉴定的酵母基因抑制 FUS 毒性的质粒转化为单倍体酵母菌株 (w303 MATα)。然后将这些酵母与 FUS 表达质粒转化为相反交配型 (w303) 的单倍体酵母交配。在含有葡萄糖 (基因 ' 脱落 ') 和半乳糖 (基因 ' 上) 的琼脂板块上, 选择了含有 FUS 的双倍酵母, 或者是一个空的载体或五抑制基因之一。(1) 用两个空载体变换了控制酵母菌株。(2) 用空载体 FUS 酵母菌株, FUS 的表达具有很大的毒性。(3–7) 显示 FUS 的双倍酵母, 以及能抢救 FUS 毒性的抑制基因。每个数字 (1–7) 显示两行十二相同的复制。这张图片, 代表三独立实验, 是在3天的增长30摄氏度后拍摄的。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig3large.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/57978/57978fig4.jpg"> 图 4: 对拯救或加剧 FUS 毒性的基因进行图书馆筛选.含有 FUS 的单倍体酵母与含有该库基因的单倍体酵母交配。交配后, 在四份中选择了含有 FUS 和库基因的双细胞, 然后发现葡萄糖 (FUS 和图书馆基因 ' 脱落 ') 和半乳糖琼脂板块 (FUS 和图书馆基因 ' on ')。在 FUS 和库基因表达的半乳糖板上, 大部分酵母不能生长良好。这表明 FUS 是有毒的, 大多数的图书馆基因无法抢救毒性。绿色广场展示了一个图书馆基因的例子, 它抑制了 FUS 的毒性, 并允许酵母形成菌落。红方表明了一个图书馆基因的例子, 加剧了 FUS 毒性。在这里显示的板块代表了10板块的图书馆基因筛选反对。这张照片是在3天的增长30摄氏度后拍摄的。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

Mating-based Overexpression Library Screening in Yeast

本文提出了一种基于交配的方法, 利用阵列质粒库, 促进萌芽酵母的超表达筛查。

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

mating-based-overexpression-library-screening-in-yeast

Nanyang Technological University

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JoVE Journal

Genetics

7.6K 次观看.  Wright State University. 本文提出了一种基于交配的方法, 利用阵列质粒库, 促进萌芽酵母的超表达筛查。

视频: Mating-based Overexpression Library Screening in Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

科研

遗传学

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

萌芽酵母 SNX-BAR 异体体的表达、纯化和脂质结合

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...