该项目的资金支助由科学和信息和通信技术部 (2016R1A2B2006354) 资助的韩国国家研究基金会 (NRF) 提供。

1. 媒体的准备工作
<ol><li>用补充剂 (是的), 没有腺嘌呤 (是低艾德), 粟谷氨酸培养基 (普纳12) 和普纳, 没有腺嘌呤 (普纳), 通过混合组件, 如表 1中所述, 准备酵母提取物。是-艾德 (低艾德) 和普纳板有所有相同的成分, 但腺嘌呤是从后者省略。<ol>
<li>使用以下盐 (50x) 库存: 52.5 克/升氯化镁2· 6H2O, 2 克/升 CaCl2· 2H2O, 50 克/升氯化钾, 2 克/升 Na2所以4在蒸馏水。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。</li>
		<li>使用以下维生素 (1,000x) 股票: 1 克/升泛酸, 10 克/升烟酸, 10 克/升肌醇, 10 毫克/升水生物素在蒸馏水。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。</li>
		<li>使用以下矿物 (10,000x) 库存: 5 克/升硼酸, 4 克/升 MnSO4, 4 克/升 ZnSO4· 7H2O, 2 克/升 FeCl2· 4H2O, 0.4 克/升 MoO3, 1 克/升, 0.4 克/升丘索4· 5H2o, 10 克/升柠檬酸在蒸馏水中。过滤消毒使用0.22 µm 孔径过滤器和存储在4摄氏度。 注: 是液体培养基可通过省略琼脂制成。</li>
	</ol>
</li>
	<li>蒸压介质, 冷却到60摄氏度以下, 搅拌时用搅拌棒, 速度为 200 rpm。</li>
	<li>对于 YES+G418 (geneticin) 板, 请将1毫升/升 G418 库存解决方案添加到 "是" 介质中。<ol>
<li>使用以下 G418 (1,000x) 库存: 100 毫克/毫升 G418 蒸馏水。过滤消毒使用0.22 µm 孔径过滤器, 整除1.5 毫升离心管, 并存储在-20 摄氏度。</li>
	</ol>
</li>
	<li>再搅拌 5-10 分钟, 将介质整除成90毫米培养皿 (中等整除数的1升至大约40培养皿。把盘子存放在4摄氏度。</li>
</ol>2. 克隆rpt4+及其 5 '/3 ' UTRs
<ol><li>用引物 p1 和 p2 (图 1A) 进行 PCR, 以裂变酵母基因组 DNA (gDNA) 作为模板, 总体积为50µL (~ 1 µg DNA, 20 U 型酶, 1x 反应缓冲器), 并应用表 2中描述的反应周期放大3315基对 (bp) 片段, 包括rpt4+及其 5 '/3 ' UTRs。使用制造商 (图 1A) 指示的纯化套件13提纯 PCR 产品。</li>
	<li>摘要 pBlueScript KS (-) 向量14和包含rpt4+的放大 DNA 片段, 其 5 '/3 ' UTRs 与BamH1和Xho1在总容积20µL (~ 1 µg DNA, 20 U 酵素, 1x 消化缓冲) 在37°c 在水浴为 16-18h (图 1B)。</li>
	<li>凝胶纯化消化载体 (1.2% 琼脂糖凝胶) 和 DNA 插入通过执行泰基琼脂糖凝胶电泳 (100 V, 1 h), 切除所需尺寸的 dna 带, 并将 dna 与凝胶萃取套件15隔离。</li>
	<li>结扎使用 T4 dna 连接酶的载体和 dna 插入, 执行 20-µL 结扎反应, 含有 50-100 ng 的载体 dna, 3 倍摩尔超过插入 dna, 400 U T4 DNA 连接酶, 1x 结扎缓冲。孵化这种混合物在18°c 16-18 小时。</li>
	<li>应用热休克七十年代42摄氏度, 将结扎后的 DNA 转化为100µL 的大肠杆菌DH5α。添加1毫升磅和孵育1小时在37摄氏度, 以恢复。执行离心 (13800 x g), 丢弃上清, 在 LB 氨苄西林 (LA) 板上全部转化的大肠杆菌细胞, 并在37摄氏度孵育16小时。</li>
	<li>选择 4-8 殖民地与牙签和增长隔夜在37°c 在3毫升 LA 媒介。</li>
	<li>根据制造商的协议, 用质粒纯化套件16隔离粒度, 并执行分析限制酶消解与5µL 的分离质粒, 5 U 的每种限制酶 (BamH1和Xho1) 和1x 限制缓冲区。在37摄氏度的水浴中孵育反应混合物3小时. 通过序列候选质粒对克隆进行确认。</li>
</ol>3. 引入无声变异 (Xho1限制网站)
<ol><li>设计一对 33-bp 引物 (p3 和 p4), 其中包含所需的突变在其他补充序列。</li>
	<li>使用表 10中列出的循环参数, 用高保真聚合酶17 (图 1C) 的总体积25µL (10 ng 克隆质粒, 2 µL 2 pmol 底漆混合, 3 U 酶, 1x 反应缓冲器) 进行 PCR。</li>
	<li>将模板质粒与DpnI 一起消化, 总容积为20µL (20 U 酵素, 1x 消化缓冲) 在37°c 在水浴中1小时。</li>
	<li>如步骤 2.5-2.7 所述, 对含有无声突变的质粒进行变换、传播、分离和序列化。 注意:这种无声突变也可以使用一种克隆方法来引入, 它允许在单个反应18中加入多个 DNA 片段。</li>
</ol>4. 随机突变的rpt4+由易出错 PCR
<ol><li>执行易出错的 PCR, 使用质粒与无声突变 (Xho1站点) 作为模板, 引物 p5 和 p6, 总容量50µL (在步骤4.1.1 定义的模板量, 2 µL 10 pmol 底漆混合, 2.5 U 酵素, 1x 反应缓冲)以及用于生成高错误率19 (图 1D) 的特殊聚合酶。按照表 2中的说明执行 PCR。<ol>
<li>使用 4-5 µg 模板质粒控制突变频率为 1 bp/kb (kilobase)。 注意:一个高浓度 (> 500 ng/µL) 的质粒, 可以获得使用迷你准备套件20, 是必需的。</li>
	</ol>
</li>
	<li>用凝胶电泳确认 2670 bp (1.2% 琼脂糖凝胶) 的 PCR 产物。凝胶纯化此 PCR 产品, 如步骤2.5 所述。</li>
</ol>5. 合成 PCR 片段 (菅直人, 3 ' UTR)
<ol><li>使用 pFA6a-KANMX621作为模板、引物 p7 和 p8, 以及表4中列出的条件来获取标记 (菅直人) 片段, 执行 PCR。凝胶纯化产生的 1480 bp 片段, 如步骤 2.5 (图 1E) 所述。<ol>
<li>检查以突变为目标的基因的停止密码子和相邻基因的编码区域是否由超过500的 bp 分离。当该区域小于 500 bp 时, 可能不使用内生终结器;相反, 应该使用抗利尿终止符而不是内生终止符。表 5中显示了使用抗利尿终结器所需的协议更改。 注意:仅当使用 pFA6a-3HA-KANMX6 质粒中可用的抗利尿终结器而不是内生终止符时, 这些更改才适用。</li>
	</ol>
</li>
	<li>用克隆的rpt4+包含的向量作为模板和引物 p9 和 p10, 在总容积50µL (20 ng 克隆质粒中) 进行 PCR, 2 µL 10 pmol 底漆混合, 2 U 型酶, 1x 反应缓冲器), 使用表 6中提供的条件。凝胶纯化获得的 506-bp 3 ' UTR 片段 (图 1E)。</li>
</ol>6. 融合 PCR 法生成转换构造 (图 1E)
<ol><li>在 50 ng/µL 上准备3个片段 (mutagenized rpt4+、菅直人和 3 ' UTR)。</li>
	<li>使用底漆 p11 和 p12 进行三片段的融合 PCR, 如表7所述。(融合 PCR 的协议是对原始协议22的修改。净化主要的 4398-bp PCR 产品。<ol>
<li>使用rpt4+: KAN:3'UTR 碎片的比率为 1:3: 1, 以获得最佳结果。 注意:插入 DNA 的总量不应超过 500 ng。建议的rpt4+数量: KAN:3'UTR 是 50 ng:150 ng:50 ng。mutagenized 区域约为 1.6 kb, 因此, 将每个基地的所有可能组合的最低数量的酵母菌落, 将被变异到其他三是 1600 x 3 = 4800 殖民地。由于一个转化反应通常产生 400-500 菌落, 至少有10反应价值的融合 PCR 结构是需要的。这种需要可以通过简单地增加反应量 (即, PCR 管的数量) 来满足, 系数为十。</li>
	</ol>
</li>
</ol>7. 用电穿孔法转化裂变酵母 (图 1F)
<ol><li>接种酵母到10毫升的是培养基饱和通过孵化超过16小时。</li>
	<li>稀释细胞到光学密度在600毫微米 (OD600) 0.2 在200毫升是中等和孵化在30°c 与震动为 5-6 h, 到 OD600 = 0.6-0.8。</li>
	<li>将单元格集中到 OD600 = 30, 免除四50毫升锥形管, 并在冰上冷却10分钟。</li>
	<li>以 1050 x g 为3分钟, 在4摄氏度进行离心, 收获细胞。把管子和10电小试管放在冰上。在冰上执行步骤 7.3-7.8。</li>
	<li>丢弃上清, 加入15毫升1.2 米山梨醇。轻轻摇动管子, 并用重悬细胞。</li>
	<li>离心细胞在 1050 x g 3 分钟在4摄氏度。</li>
	<li>重复步骤7.4 和7.5。放弃上清。添加500µL 1.2 米山梨醇的每管, 并用重悬细胞, 并收集所有的细胞在一个15毫升锥管。</li>
	<li>添加1.2 米山梨醇高达2.4 毫升 (OD600 = 10 每0.2 毫升)。把管子放在冰上。</li>
	<li>添加200µL 山梨醇悬浮细胞 (确保它们完全悬浮) 到含有融合 PCR 结构的 EP 管, 混合好, 并将样品转移到电试管。</li>
	<li>Electroporate 细胞具有以下选择: 真菌, ShS (2.00kV, 1 脉冲)。</li>
	<li>添加600µL 1.2 米山梨醇对每个电试管的总容量800µL, 然后传播细胞在四是板材 (200 µL/板材)。十电小试管将免除40是板材。</li>
	<li>在30摄氏度孵化出24小时的板材。</li>
	<li>对 YES+G418 进行复制, 并在30摄氏度的3天内孵化板材。</li>
</ol>8. 染色质不稳定突变体的选择和检查误报
<ol><li>对于每个 YES+G418 板, 执行副本电镀, 以是艾德 (低艾德) 和普纳 (无艾德) 板。将复制板孵化 1-2 天, 在30摄氏度, 直到一些在 YES 板上的殖民地显示红色着色 (图 1G)。</li>
	<li>比较是艾德和普纳板, 并选择显示粉红色或白色的在 "是" 板上, 也生长在普纳板上的细胞。不要在普纳上选择没有生长的菌落, 因为它们是假阳性 (例如,反映了菅直人在基因组其他地方的非目标站点被集成)。</li>
	<li>从每个菌落中选取大约 1 x 105单元格, 并在10µL 的 SPZ 溶液中孵化, 其中包含2.5 毫克/毫升 zymolyase 100 T, 37 摄氏度, 至少30分钟. 使用此解决方案的1µL 作为菌落 PCR (图 1H) 的起始模板执行。<ol>
<li>使 SPZ (50 毫升) 混合30毫升2米山梨醇, 4.05 毫升1米 Na2HPO4, 0.95 毫升 NaH2PO4和15毫升蒸馏水 (最终 pH, 7.5)。样品 (5 毫升) 可以补充与 zymolyase 和存储在 500-µL 整除数在-20 °c, 直到使用。</li>
	</ol>
</li>
	<li>进行凝胶电泳 (1.2% 琼脂糖凝胶, 180V, 20 分钟), 以可视化的结果 + 菌落 PCR。选择与XhoI (4 U 酵素, 1x 消化缓冲, 37 °c 水浴, 1 h) 一起展示适当的大小的 PCR 带和消化5µL 的反应产品, 以筛出误报 (图 1H)。</li>
	<li>进行凝胶电泳以可视化XhoI文摘 (1.2% 琼脂糖凝胶, 180 V, 20 分钟)。标记其 PCR 产品由XhoI剪切的菌落, 并将它们贴到 YES+G418 板上 (图 1I)。</li>
	<li>将 gDNA 与裂变酵母细胞分离, 并执行 PCR 产品的序列化23。将获得的序列与野类型序列进行比较, 以识别突变 (图 1I)。</li>
	<li>通过将突变细胞的 PCR 扩增的融合结构转化为野生型细胞, 直接将突变引入23。使用斑点来确认新制作的突变细胞中的表型 (图 1J)。<ol>
<li>利用突变体的基因组 DNA 作为模板, 在50µL (~ 1 µg DNA、20 p1 酶、1x 反应缓冲器) 中, 通过 PCR 与引物和 p10, 可以很容易地扩增融合转化结构, 并应用表2中描述的反应周期. .构造的转换可以通过以下步骤 7.1-7.13 完成。</li>
	</ol>
</li>
</ol>

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D., 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+19S+proteasome+is+directly+involved+in+the+regulation+of+heterochromatin+spreading+in+fission+yeast.">The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast.</a> J Biol Chem. , (2017).</li><li>Tang, N. H., 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=Generation+and+characterisation+of+temperature+sensitive+mutants+of+genes+encoding+the+fission+yeast+spindle+pole+body.">Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body.</a> PeerJ Preprints. 5, e3377v3371 (2017).</li><li>Rasooly, A., Weisz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibacterial+activities+of+phloxine+B+and+other+halogenated+fluoresceins+against+methicillin-resistant+Staphylococcus+aureus.">In vitro antibacterial activities of phloxine B and other halogenated fluoresceins against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 46 (11), 3650-3653 (2002).</li><li>Wilson, D. S., Keefe, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+mutagenesis+by+PCR.">Random mutagenesis by PCR.</a> Curr Protoc Mol Biol. , (2001).</li></ol>

作者没有什么可透露的。

随机突变通过容易出错的 PCR 是一个强大的工具, 以产生一个不同的突变池在某一地区。这种技术对于那些试图在特定情况下评估蛋白质功能的研究尤其有用。例如, 我们在这里使用了易出错的 PCR 评估19S 蛋白酶体亚基, Rpt4, 在染色质维护的功能。通过改变容易出错的 PCR 和调整筛选条件的区域, 我们能够在感兴趣的基因组区域和屏幕上变异细胞, 以期望的表型。最近, 一种类似的方法被用来隔离在主轴杆体组件25中窝藏突变的裂变酵母细胞。
随机诱变的关键优势在于, 它可以应用于非必需基因和基本遗传因子。随机突变的基本基因可能产生多样的突变体, 如那些微妙和/或部分功能缺陷, 允许细胞生存能力, 或变种人的功能只受特定条件的影响。后者的一个例子是温度敏感突变体。这种突变体可能被隔离使用5毫克/升 phloxine B, 它用红色染色死亡细胞, 使缓慢生长的细胞与垂死的细胞26区分开来。
该协议的关键步骤是易出错的 PCR 步骤。在这一步骤中, 控制突变频率是很重要的, 这取决于 PCR 周期的数量和模板的初始量。我们建议用户修复 PCR 周期的数量, 并改变初始数量的模板, 因为我们发现这是一个更方便的优化策略。我们注意到, 容易出错的 PCR 技术已经在几十年里得到了广泛的应用。如果用户选择, 他们可能会寻求一种方法, 以手动执行易出错的 PCR27 , 而不使用商业上可用的诱变工具包。
谨慎地说, 这个协议的假阳性率相当高。因此, 变种候选者应该进行一系列的筛查, 以消除误报。我们发现, 在目标基因中无声地纳入限制站点可以帮助避免将误报标入相对费力的排序步骤的需要。这是成本效益, 因为突变的候选人可能在数以百计甚至更远的数字。

前遗传学是一种经典的方法, 研究人员寻找自然发生的突变体, 显示特定的表型, 并进行遗传分析。在反向遗传学中, 突变被引入到一个感兴趣的基因中, 并检查表型。在后一种情况下, 目标基因的随机突变经常被用来产生一个突变体, 随后被选择为期望的表型, 如温度敏感性或改变的酶活性。各种方法可用于实现随机突变, 包括易出错的 PCR1;紫外线照射2;化学诱变, 如甲基磺酸乙酯 (EMS) 或一氧化二氮3;使用临时突变菌株, 如过度表达mutD5 4;和 DNA 洗牌5。
在这里, 我们描述了一个反向遗传学策略, 我们利用易出错的 PCR 产生突变池的目标基因的裂变酵母。从它的名字可以推测, 这种方法通过在 PCR 过程中故意引入错误来产生突变。与其他突变方法不同, 容易出错的 PCR 允许用户定义要 mutagenized 的区域。这使得它特别有用的努力研究的功能蛋白质/领域的兴趣。
为了演示这个随机的突变过程, 我们在这里使用rpt4+, 它编码19S 蛋白酶体亚基, 作为一个例子。Rpt4 在裂变酵母6、7、8、9之外的有机体中已被证明具有与蛋白质分解无关的功能, 而蛋白质水解的缺陷可能通过改变蛋白而导致间接影响。水平。因此, 我们对突变体进行了筛选, 从而引发了与蛋白水解无关的变化, 目的是研究蛋白酶体对染色质的作用。
通过调整引物的位置, 可将易出错的 PCR 应用于任何基因区域。表现出期望表型变化的突变体可以与适当的记者鉴别。在这里, 我们使用了插入到着丝粒1外部重复 (工程机械)区域10的ade6+ 记者。本构染色质是在该区域11上形成的, 因此在野外类型条件下, ade6+报告器处于静音状态;红色殖民地10表示这一点。在着丝粒上破坏本构染色质的突变将导致ade6+ 的表达式, 该报告被可视为白色的菌落。

<table border="0" cellpadding="0" cellspacing="0" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1. Media</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Glucose</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-10KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Yeast extract</td>
			<td style="width:155px;">BD Biosciences</td>
			<td style="width:119px;">212720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">L-Leucine</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">87070-0310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Adenine sulfate</td>
			<td style="width:155px;">ACR</td>
			<td style="width:119px;">16363-0250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Uracil&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">U0750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">L-Histidine</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">KH2PO4</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">1.05108.0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">NaCl</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">19015-0350</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">MgSO4&#8226;7H2O</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">63140</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">CaCl2</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">12095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Potassium phthalate</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6758-500g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Inositol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7508-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Biotin</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">B4501-100MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Boric Acid</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">B6768-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MnSO4</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7634-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">ZnSO4&#8226;7H2O&#160;</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">83060-031</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">FeCl2&#8226;4H2O</td>
			<td style="width:155px;">KANTO</td>
			<td style="width:119px;">CB8943686</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Sodium molybdate dihydrate</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">31621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">KI</td>
			<td style="width:155px;">JUNSEI</td>
			<td style="width:119px;">80090-0301</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">CuSO4&#8226;5H2O</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">09605</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">D-myo-inositol</td>
			<td style="width:155px;">MP biomedicals</td>
			<td style="width:119px;">102052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Pantothenate</td>
			<td style="width:155px;">YAKURI</td>
			<td style="width:119px;">26003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Nicotinic Acid</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">N4126-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:267px;">(NH4)2SO4&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4418-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Agarose</td>
			<td style="width:155px;">Biobasic</td>
			<td>D0012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">G418, geneticin</td>
			<td style="width:155px;">LPS</td>
			<td style="width:119px;">G41805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">2. Enzyme reactions</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">PfuUltra II Fusion HS DNA Polymerase</td>
			<td style="width:155px;">Aglient</td>
			<td style="width:119px;">600380</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">GeneMorphII Random mutagenesis Kit</td>
			<td style="width:155px;">Aglient</td>
			<td style="width:119px;">200500</td>
			<td>For error-prone PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:155px;">Thermo Fisher</td>
			<td>F-530L</td>
			<td>For fusion PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Ex Taq&#160;DNA Polymerase</td>
			<td style="width:155px;">Takara</td>
			<td style="width:119px;">RR001b</td>
			<td>For general PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">BamH1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td style="width:119px;">R0136S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Xho1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Dpn1</td>
			<td style="width:155px;">New England BioLabs</td>
			<td style="width:119px;">R0176S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">3. Equipment</td>
			<td style="width:155px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Velveteen square, black</td>
			<td style="width:155px;">VWR</td>
			<td style="width:119px;">89033-116</td>
			<td>For replica</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Replica-plating tool</td>
			<td style="width:155px;">VWR</td>
			<td style="width:119px;">25395-380</td>
			<td>For replica</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MicroPulser Electroporator</td>
			<td style="width:155px;">Biorad</td>
			<td style="width:119px;">1652100&#160;</td>
			<td>For fission yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Electroporation Cuvettes, 0.2 cm gap</td>
			<td style="width:155px;">Biorad</td>
			<td style="width:119px;">1652086&#160;</td>
			<td>For fission yeast transformation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Thermal Cycler</td>
			<td style="width:155px;">Bioer</td>
			<td style="width:119px;">BYQ6078</td>
			<td>For fusion PCR ramp reaction&#160;</td>
		</tr>
	</tbody>
</table>

gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast

随机突变的目标基因通常是用来确定突变, 产生预期的表型。在可用于实现随机诱变的方法中, 容易出错的 PCR 技术是一种方便而有效的策略, 用于生成多样性的突变体 (i. e, 一个变种人库)。容易出错的 PCR 是一种选择的方法, 当研究人员试图变异一个预先定义的区域, 如编码区域的基因, 而离开其他基因组区域不受影响。突变体库通过易出错的 PCR 扩增后, 必须克隆成合适的质粒。由于克隆步骤的效率的限制, 容易出错的 PCR 所产生的库的大小受到制约。然而, 在裂变酵母中,粟粟中, 克隆步骤可以用高效的一步融合 PCR 来代替, 从而产生转化的构造。然后, 可以使用适当的记者选择所需表型的突变体。在这里, 我们详细描述了这个策略, 以一个例子, 一个记者插入到 centromeric 染色质。

通过遵循图 1中描述的过程, 可以通过评估菌落的颜色来分析获得的 Rpt4 突变体。在减少图 2中的单元格数时, 会在相关的板块上发现菌落的颜色。插入到染色质区域的ade6+报告器在野生类型中被静音, 并显示在 "YES" 板块中的红色菌落。一旦染色质被破坏, 并且ade6+报告器被表达, 可以在 "YES" 板块中观察到白色的菌落, 如clr4Δ变种。筛选出的 Rpt4 突变体如图所示。rpt4-1突变体显示最严重的染色质不稳定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57499/57499fig1.jpg"> 图 1: 协议的示意图表示形式。(A)第一轮 pcr 所获得的 pcr 产品的示意图表示法, 它是用引物1和2进行的。(B)基于限制的rpt4+片段的克隆。(C)对克隆的向量进行站点定向诱变, 用于引入一个静默变体, 该突变添加了XhoI限制站点。(D) rpt4+编码区域的随机突变是使用易出错的 PCR 方法进行的。(E)突变的rpt4+片段、菅直人片段和 3 ' UTR 片段通过融合 PCR 连接, 生成一个转换就绪的卡带。(F)裂变酵母细胞与融合 PCR 构造相转化, 用同源重组取代内源性rpt4+序列。(G)菅直人选择的殖民地是被镀到 "是" (低艾德) 和普纳 (无艾德) 板块的复制品, 并选择了阳性菌落。(H) pcr 和随后的XhoI限制 pcr 产品用于避免误报。(I)通过对所选菌落的修补、细胞的传播、基因组 DNA (gDNA) 的提取以及rpt4+相关部分的排序, 来确定导致表型的突变。(J)通过直接将突变引入到野生型细胞中, 证实了病因突变 (并排除了误报)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57499/57499fig1large.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/57499/57499fig2.jpg"> 图 2:染色质抑制 Rpt4 突变体.otr1::ade6+记者 (top) 的示意图。以染色质失稳 (下) 为稀释, 选取筛选出的 Rpt4 突变体的5倍串联序列。这个数字被修改了从文章 ' 19S 蛋白酶体直接地参与在分裂酵母的染色质传播的章程由 Seo et, 201724。未裁剪的图形可以在原始文章中找到。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57499/57499fig2large.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 rowspan="2">组件</td>
			<td colspan="2">板材类型</td>
		</tr>
<tr>
<td>是的</td>
			<td>普纳</td>
		</tr>
<tr>
<td>酵母提取物</td>
			<td>5克/升</td>
			<td></td>
		</tr>
<tr>
<td>葡萄糖</td>
			<td>30克/升</td>
			<td>20克/升</td>
		</tr>
<tr>
<td>腺 嘌呤</td>
			<td>0.15 克/升</td>
			<td>0.1g/升</td>
		</tr>
<tr>
<td>三联</td>
			<td>0.15 克/升</td>
			<td>0.1g/升</td>
		</tr>
<tr>
<td>亮 氨 酸</td>
			<td>0.15 克/升</td>
			<td>0.1g/升</td>
		</tr>
<tr>
<td>尿 嘧啶</td>
			<td>0.15 克/升</td>
			<td>0.1g/升</td>
		</tr>
<tr>
<td>氢 pthalate 钾</td>
			<td></td>
			<td>3克/升</td>
		</tr>
<tr>
<td>Na2HPO4
</td>
			<td></td>
			<td>2.2 克/升</td>
		</tr>
<tr>
<td>l-谷氨酸</td>
			<td></td>
			<td>3.75 克/升</td>
		</tr>
<tr>
<td>盐</td>
			<td></td>
			<td>20毫升/升</td>
		</tr>
<tr>
<td>维生素</td>
			<td></td>
			<td>1毫升/升</td>
		</tr>
<tr>
<td>矿物</td>
			<td></td>
			<td>0.1 毫升/升</td>
		</tr>
<tr>
<td>琼脂</td>
			<td>16克/升</td>
			<td>16克/升</td>
		</tr>
</tbody></table>表1。是和普纳板的组分
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>周期 (s)</td>
		</tr>
<tr>
<td>95oC</td>
			<td>2分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>95oC</td>
			<td>二十年代</td>
			<td rowspan="3">30个周期</td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
		</tr>
<tr>
<td>72oC</td>
			<td>2分钟</td>
		</tr>
<tr>
<td>72oC</td>
			<td>8分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>8oC</td>
			<td>举行</td>
			<td></td>
		</tr>
</tbody></table>表2。现场定向诱变 PCR 方案推荐
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>周期 (s)</td>
		</tr>
<tr>
<td>95oC</td>
			<td>2分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>95oC</td>
			<td>三十年代</td>
			<td rowspan="3">19个周期</td>
		</tr>
<tr>
<td>55oC</td>
			<td>1分钟</td>
		</tr>
<tr>
<td>72oC</td>
			<td>7分钟</td>
		</tr>
<tr>
<td>72oC</td>
			<td>10分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>8oC</td>
			<td>举行</td>
			<td></td>
		</tr>
</tbody></table>表3。推荐的 pcr 程序为易出错 pcr
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>周期 (s)</td>
		</tr>
<tr>
<td>95oC</td>
			<td>2分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>95oC</td>
			<td>二十年代</td>
			<td rowspan="3">30个周期</td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
		</tr>
<tr>
<td>72oC</td>
			<td>2分钟</td>
		</tr>
<tr>
<td>72oC</td>
			<td>8分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>8oC</td>
			<td>举行</td>
			<td></td>
		</tr>
</tbody></table>表4。获得菅直人片段的推荐 PCR 方案
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>改变</td>
			<td>自</td>
			<td colspan="2">rpt4+ 例</td>
		</tr>
<tr>
<td>模板向量</td>
			<td>pFA6a-3HA-KANMX6</td>
			<td colspan="2"></td>
		</tr>
<tr>
<td>p7 的矢量结合序列</td>
			<td>ATTACGCTGCTCAGTGCTGA</td>
			<td colspan="2" rowspan="2">ttgctgacctgaagaaacttgaaggtacaattgattaccaaaagctttag ATTACGCTGCTCAGTGCTGA</td>
		</tr>
<tr>
<td>p7 悬挂序列</td>
			<td>最后的50bp 序列, 包括停止密码子</td>
		</tr>
<tr>
<td>p8 悬挂序列</td>
			<td>在停止密码子 (互补序列) 之后的第一个50bp 序列</td>
			<td colspan="2">AATCTTCATCGGTAAACTTATCATTTCATGGCTTTTTGGATATATGTGCA GAATTCGAGCTCGTTTAAAC</td>
		</tr>
<tr>
<td>p9 序列</td>
			<td>开始右后停止密码子</td>
			<td colspan="2">tgcacatatatccaaaaagccatgaa</td>
		</tr>
<tr>
<td>p10 序列</td>
			<td>用 p9 (互补 seqeucne) 生产 500 bp 碎片</td>
			<td colspan="2">TAGACGTTTTTCCTCGTTTCTTTGTC</td>
		</tr>
</tbody></table>表5。使用抗利尿终止符而不是内生终结器时所需的更改
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>周期 (s)</td>
		</tr>
<tr>
<td>95oC</td>
			<td>2分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>95oC</td>
			<td>二十年代</td>
			<td rowspan="3">30个周期</td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
		</tr>
<tr>
<td>72oC</td>
			<td>1分钟</td>
		</tr>
<tr>
<td>72oC</td>
			<td>8分钟</td>
			<td>1周期</td>
		</tr>
<tr>
<td>8oC</td>
			<td>举行</td>
			<td></td>
		</tr>
</tbody></table>表6。推荐 PCR 程序获得 3 ' UTR 片段
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>斜坡</td>
			<td>周期 (s)</td>
		</tr>
<tr>
<td>94oC</td>
			<td>2分钟</td>
			<td></td>
			<td>1周期</td>
		</tr>
<tr>
<td>94oC</td>
			<td>二十年代</td>
			<td></td>
			<td rowspan="4">10个周期</td>
		</tr>
<tr>
<td>70oC</td>
			<td>1 s</td>
			<td></td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
			<td>0.1 oc/s</td>
		</tr>
<tr>
<td>68oC</td>
			<td>两点半分钟</td>
			<td>0.2 oc/s</td>
		</tr>
<tr>
<td>94oC</td>
			<td>二十年代</td>
			<td></td>
			<td rowspan="4">5个周期</td>
		</tr>
<tr>
<td>70oC</td>
			<td>1 s</td>
			<td></td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
			<td>0.1 oc/s</td>
		</tr>
<tr>
<td>68oC</td>
			<td>两点半分钟</td>
			<td>0.2 oC/秒 + 5 s/周期</td>
		</tr>
<tr>
<td>94oC</td>
			<td>二十年代</td>
			<td></td>
			<td rowspan="4">10个周期</td>
		</tr>
<tr>
<td>70oC</td>
			<td>1 s</td>
			<td></td>
		</tr>
<tr>
<td>55oC</td>
			<td>三十年代</td>
			<td>0.1 oc/s</td>
		</tr>
<tr>
<td>68oC</td>
			<td>两点半分钟</td>
			<td>0.2 oC/秒 + 二十年代/周期</td>
		</tr>
<tr>
<td>72oC</td>
			<td>10分钟</td>
			<td></td>
			<td>1周期</td>
		</tr>
<tr>
<td>8oC</td>
			<td>举行</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表7。融合 pcr 的推荐 pcr 方案
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr>
<td>CBL1877</td>
			<td>h +</td>
			<td>ade6-210 leu1-32 ura4-D18 otr1R (dg 谷氨酸) Sph1:ade6</td>
		</tr></tbody></table>表 S1: 本研究中使用的应变
<img src="/files/ftp_upload/57499/57499tbl2.jpg"> 表 S2: 本研究中使用的引物列表

Watch this Scientific Journal Video about Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast at JoVE.com

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

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

本文介绍了一种用于裂变酵母靶基因随机诱变的详细方法。例如, 我们针对的是rpt4+, 它对19S 蛋白酶体的亚基进行编码, 并筛选出破坏染色质的突变。

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 ...

gene-targeted-random-mutagenesis-to-select-heterochromatin

Nanyang Technological University

Research

JoVE Journal

Genetics

10.6K 次观看.  Korea Advanced Institute of Science and Technology. 本文介绍了一种用于裂变酵母靶基因随机诱变的详细方法。例如, 我们针对的是rpt4+, 它对19S 蛋白酶体的亚基进行编码, 并筛选出破坏染色质的突变。

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

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 ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

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 ...

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 ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...