这项工作是由美国国防部高级研究计划局的合同N66001-12-C-4039支持YS，RSL，Alfred P. Sloan基金会的资助和美国国立健康补助R01 HG003224和R21 CA130266 FPRFPR也从加拿大高级研究所的奖学金，由加拿大卓越研究教席计划的支持。

1。生成ProMonsters <ol><li>准备通用GFP替换盒扩增TETO 2-GFP标记和URA3标记从质粒pYOGM012的（氨苄青霉素抗性）使用引物序列： ：GGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCC CAAGCTCCTCGAGTAATTCG和GGCGTTAGTATCGAATCGACAGCAGTATAGCGACCAGCATTCAC GTACCGGGTAATAACTGATATAAT（粗体地区提供磁带允许的KanMX4有针对性的更换侧翼区的同源性）。应进行的PCR反应Phusion聚合酶，HF缓冲液，和3％的二甲亚砜（New England Biolabs公司）与模板DNA浓度为0.5 ng /μl的开始。 PCR程序为98℃30秒，35个循环的98℃，持续10秒，49℃下30秒，和72℃1.5分钟。反应总体积50μl到每个转换实验提供足够的DNA。我们净化使用QIAquick PCR纯化试剂盒（Qiagen公司）的产品与〜50微升的PCR反应，每列处理，但跳过的纯化步骤可能会增加转化体的产率。 另外，释放的片段，含有2-GFP的TETO和URA3标记，以及同源区KanMX4的定位中，通过使用的限制酶EcoRI（New England Biolabs公司）消化质粒pYOGM057个（氨苄青霉素抗性）。在该反应中的DNA浓度应为〜30纳克/微升。反应总体积34μL。 对于集成的GFP的盒式录像带插入的区域以外KanMX4，调整上面的相应的同源序列的PCR引物的同源性区域。 </li><li>变换的普遍GFP缺失盒与一个单倍体的应变从该KanMX4的酵母基因敲除收集12。伍兹和Gietz 13 0.34μl的协议DNA样品（纯化的PCR产物或非纯化的限制性内切酶消化）应该用于每个变换实验。板的五分之一的转化混合物到民航局-URA板（0.67％酵母氮​​基，2％葡萄糖，0.6％酪蛋白氨基酸，腺嘌呤硫酸盐0.0025％，0.005％色氨酸，和2％琼脂）。 </li><li>数列转化上一个新的CAA-市建局板获得分离菌落。 </li><li>细胞从一个殖民地转移到YPDA板含有200μg/ ml的G418确认缺乏增长。确认成功GFP融合，进行菌落PCR步骤（1.5） - （1.9）。 </li><li>添加取自菌落到2微升的裂解缓冲液（0.1M磷酸钠，pH值7.4，1的单元ZYMOLYASE从ZymoResearch）在0.2毫升的PCR管或96孔板的孔中的细胞。混合，通过移液的溶液中的移液管尖，满分。 </li><li>覆盖与一滴（约20微升）的矿物油使用P200 PIPETMAN（吉尔森）。 </li><li>在37℃孵育混合物C为20分钟，然后在95℃下进行10分钟。 </li><li>稀释的溶胞产物用50μl水。混合该溶液通过移液和10倍。 </li><li> 1微升的溶胞产物与（A）退火上游侧翼区的正向引物配对的引物的序列CCTGAGAAAGCAACCTGACC（285 bp的从一开始的磁带盒）使用10微升的PCR反应，（B）一种反向分析引物退火到配对的引物的序列GCATTGGGTCAACAGTATAG（375 bp的从端部）的下游区域，（C）的两个引物退火到KanMX4与序列CGTACTCCTGATGATGCATG和GACGAAATACGCGATCGCTG（181 bp）的，和（D）的两个引物退火的靶基因（ 图4）的野生型序列。 （D）是很重要的，因为在删除集合约8％的菌株是已知的含有非整倍体14。正确的转化体应该是正与（A）和（B），和负的（C）和（D）。 </li><li>要引入到的转化体的GMToolkit成1.6-ml Eppendorf管中，添加大约250,000细胞从转化菌株和250000细胞的任一RY0146（含有GMToolkit-α）或RY0148（含有GMToolkit-α）。涡街流量计。 RY0146和RY0148的基因型是MATαlyp1Δhis3Δ1leu2Δ0ura3Δ0met15Δ0can1Δ:: GMToolkit 一个 [CMVpr rtTA KanMX4 STE2pr SP-his5的]和MATαlyp1Δhis3Δ1leu2Δ0ura3Δ0met15Δ0can1Δ:: GMToolkit-α[CMVpr rtTA NatMX4 STE3pr LEU2。 Pr表示启动。细胞数，应当估计前述文化从光学密度。 </li><li>离心800×g离心2分钟。 </li><li>除去上清液。 </li><li>为了允许空气交换，使使用图钉用70％乙醇处理的盖子上的孔。 </li><li>加入50μlYPDA培养基（1％酵母提取物，2％蛋白胨，2％葡萄糖，0.003％腺嘌呤hemisulfatE）。涡街流量计。 </li><li> 800×g离心5分钟离心。 </li><li>将管在湿布框中。这可以创建一个空的提示框，一个小技巧的立场，并用湿纸巾。 </li><li>过夜孵育箱30°C。 </li><li>选择交配二倍体第一个裸奔的细胞在CAA浦板。板在30℃温育一天。 </li><li>以细胞从本体区域，条纹出来，使分离的集落YPDA板（1％酵母提取物，2％蛋白胨，2％葡萄糖，0.003％腺嘌呤半硫酸盐，和2％琼脂），含有200微克/毫升的G418选择二倍体含有GMToolkit-a或100微克/毫升nourseothricin（纳特）15选择二倍体含有GMToolkit-α的。 </li><li>板在30℃温育3天。 </li><li>从开始一个孤立的菌落，大多数的细胞转移至500微升GNA介质没有琼脂（1％酵母提取物，3％Difco公司营养肉汤，和5％葡萄糖，高压釜中的葡萄糖和其余的t他组件分别为2×的解决方案，以避免焦糖）在5毫升的培养管与盖松开。 </li><li>水平旋转管五个小时，在30℃下旋转的轴线的需要是在管的纵向轴线平行。 </li><li>确认所用的殖民地（1.21）市建局+的裸奔出来在CAA浦板。 </li><li>离心管中于800×g离心2分钟。弃上清。 </li><li>加入500μl的最小产孢培养基（1％的醋酸钾，0.005％醋酸锌;过滤灭菌）。涡街流量计。 </li><li>离心管中于800×g离心2分钟。弃上清。 </li><li>重复（1.25） - （1.26）的两倍。 </li><li>加入1毫升补充有7.5微克/毫升赖氨酸，7.5微克/毫升5微克/毫升组氨酸，亮氨酸，5微克/毫升的蛋氨酸和1.25微克/毫升尿嘧啶（提高的营养缺陷型菌株的孢子形成率的最小的产孢培养基） 。涡街流量计。 </li><li>旋转管，在室温下为一天。 </li><li>旋转管，在30℃，2天。 </li><li>离心机125微升该混合物在1.6-ml Eppendorf管中，在800×g离心2分钟。弃上清。 </li><li>加入50μl的ZYMOLYASE液（100mM磷酸钠缓冲液，pH值7.4，1 M山梨糖醇，和2单位ZYMOLYASE从ZymoResearch）。混合，通过移液的溶液中的移液管尖，满分。 </li><li>在30℃孵育1小时。 </li><li>加入50μl0.02％NP-40。混合，通过移液的溶液中的移液管尖，满分。 </li><li>将离心管在室温下五分钟。 </li><li>加入500μl的水。 </li><li>管放置在冰上。 </li><li>超声波清洗两次这种混合物在输出设置为1（最弱的设置），使用3.2毫米的微尖SONIFIER 450（布兰森），持续15秒。在两轮之间的超声，把管上的冰〜0°C的温度返回到</li><li>离心管，在800×g离心5分钟。弃上清。 </li><li>加入200μlSC​​-URA-（a）或SC-URA-亮氨酸（α）的介质，这取决于是否交叉为纳入GMToolkit-a或GMToolkit-α。要准备媒体，使SC-URA-的His-亮氨酸培养基（0.67％酵母氮​​基，2％葡萄糖，0.01％的精氨酸，天门冬氨酸0.01％，0.01％的谷氨酸，0.01％异亮氨酸，0.01％赖氨酸，0.01％蛋氨酸，苯丙氨酸0.01％，0.08％，丝氨酸，0.04％的苏氨酸，0.002％酪氨酸，0.03％缬氨酸，0.0025％腺嘌呤，0.005％色氨酸，0.003％腺嘌呤，0.005％色氨酸）和补充用无菌组氨酸或亮氨酸的使用前的最终浓度为0.01％。 </li><li>请使用图钉用70％乙醇处理的盖子上的孔。 </li><li>水平旋转管在30℃，2天。旋转的轴线的需要是在管的纵向轴线平行。 </li><li>出的细胞上的SC-URA-His或SC-URA-Leu取代琼脂平板上（与上述相同的成分加2％琼脂，苏氨酸和天门冬氨酸的连胜后，必须添加utoclaving添加组氨酸，亮氨酸在浇注前）的ProMonster株的分离菌落。 </li></ol> 2。性别骑自行车<ol><li>旋转一个既定的GFP的缺失在100μl的SC-His的（a）或SC-亮氨酸（α）在30取决于交配型菌株的培养℃下保温过夜使用了1.6-ml Eppendorf管中。进行空气交换，使一个洞，用70％的乙醇使用图钉。水平旋转管。旋转的轴线的需要是在管的纵向轴线平行。它可以跨越多株，通过集体交配。当一个排序的试样直接用于这个目的，培养细胞在30℃在200μl2天。为了制备介质，SC-URA-His的亮氨酸培养基中添加尿嘧啶（终浓度：0.0025％）和组氨酸（0.01％）或亮氨酸（0.01％）。 </li><li>在1.6毫升EPPE单倍体细胞250,000以及250,000的α-单倍体细胞的GFP缺失菌株混合ndorf管。涡街流量计。 </li><li>交配这些细胞，按下面的步骤（1.11） - （1.17）。 </li><li>配合混合物10微升转移到500μlGNA中的5毫升的培养管中的介质含有200微克/毫升的G418和100微克/毫升纳特松开盖。 OD 600起大约是0.1。 </li><li>在30°C，24小时旋转的文化。这允许为二倍体的选择，因为只有在二倍体包含两个KanMX4的 GMToolkit-，a和NatMX4在GMToolkit-α可以生长在G418和Nat的存在（ 图3）。 </li><li> 20微升的为期一天的文化转移到500μl新鲜的GNA含G418和NAT的。 OD 600起大约是0.2。 </li><li>旋转管在30℃，5小时，使细胞对数生长期（OD 600为〜1）。 </li><li>按照下列步骤（1.24） - （1.38）形成孢子的二倍体和隔离孢子。 </li><li>暂停孢子化的细胞分割成两个300微升份，并把每一个到一个单独的1.5 ml Eppendorf管中。 </li><li>离心管，在800×g离心5分钟。弃上清。 </li><li>一个试管中的沉淀，加入100μl的SC-他的媒介选择单倍体。其他管沉淀，加入100μlSC​​-Leu的培养基中，选择α-单倍体。 </li><li>旋转管于30℃下过夜。最终OD 600应该是小于0.5。如果OD 600会变得过高的倾向，尝试在室温下培养。 </li><li>为了诱导GFP，加入100μl新鲜的SC-（a）或SC-亮氨酸（α）中含20微克/毫升强力霉素。多西环素的最终浓度是10微克/毫升。涡街流量计。 </li><li>旋转管在30℃下2天。 </li></ol> 3。流式细胞仪<ol><li>加入900μl的预滤波的TE缓冲液（pH 7.5）中含10微克/毫升多西环素的5毫升的聚丙烯管（BD猎鹰＃352063），与帽交换与细胞从5毫升的聚苯乙烯管滤网盖（BD猎鹰＃352235）。聚丙烯管中是适合流式细胞仪分析。需要去除大颗粒滤网盖。 </li><li>轻轻地放置75微升TE缓冲液中，用强力霉素细胞滤网盖。 </li><li>帽上的诱导后的细胞的溶液中加入25μl。 </li><li>虽然按提示PIPETMAN对网格，拍摄的联合解决方案通过过滤器。 </li><li>按下滤网盖和涡管。 </li><li>准备填充用200μl的SC-His或SC-Leu的收集管（1.6毫升Eppendorf管中）。 </li><li>启动细胞分选和调整设置，根据制造商手册。 </li><li>涡收集管涂在墙上介质和管含有细胞，然后再装入到细胞分选仪。 </li><li>采集的样品的流式细胞术数据。不含有GFP的应变是一种消极的控制。 </li><li>绘制门进行排序。 （A）为了避免细胞聚集体，具有较高的GFP强度，丢弃的离群值不成比例的大脉冲宽度，前向散射（FSC）FSC高度和FSC的阴谋阴谋的脉冲宽度和脉冲高度MoFlo分拣机或小得不成比例FSC高度FACSAria分拣机的区域（ 图5，顶左面板）。 （B）由于大的FSC（地区）的预期的细胞聚集体，只需要在FSC的低20％的细胞中，同时避免与最低FSC和侧向散射（SSC）的值，其中的细胞碎片常常发现的区域（ 图5中 ，右上角面板）。 （C）SSC（区）和GFP信号（区）往往呈正相关。因为简单地把给定上面的栅极最亮的细胞也将丰富细胞与高SSC值，绘制的人口的周边使用的曲线图的GFP强度和SSC采​​取类似的百分比的最亮的细胞从每个点的栅极沿着的SSC的轴（ 图5，博特OM-左图）。在（C）中选择的人口，应该是0.1-1％的（B）中选择的人口。 </li><li> 200个细胞分类收集管中的标准（3.10）。对于一个集体过程或其他项目需要一个更复杂的人口，更多的细胞进行排序。 </li><li>涡收集管细胞中等。 </li><li>板上YPDA板用于基因分型的细胞的一个子集（例如，50个细胞）。 </li><li>板在30℃孵育2天。 </li><li> 〜12个殖民地的步骤（1.5） - （1.8）的裂解物。 </li><li>的残余细胞的尖端（1.5）到的光斑在YPDA板的G418和Nat用刀尖轻轻抚摸板。选择单倍体，不应该让这个板块。二倍体可能有更多的绿色荧光蛋白的副本，因此可能是美好的。这个测试可以显示单倍体选​​择的效率。 </li><li>用于基因分型，1微升溶胞产物进行分析，用10微升的PCRŕeaction用的正向引物，退火上游侧翼区的每个已删除的基因的引物的序列CCTGAGAAAGCAACCTGACC搭配。 （如果可能的多重PCR反应条件，可确定使用的单突变体的裂解）。 PCR可以在96 - 或384孔的格式。后者的格式是适用于低至5微升的反应体积。阵列PCR主混合不同的引物和这些混合物的细胞裂解物的添加，可以进行使用一个多通道移液管或液体处理机器人。提示的变化是可选的，不同的PCR混合时加入裂解液。 </li><li>分析PCR产物。凝胶XL超V-2电泳系统（Labnet），是装样使用多道移液器兼容。 </li><li>基于信息（3.16）和（3.18），所需的基因型，确定可能的单倍体。 </li><li>建立一个新的板（YPDA，CAA，市建局，或SC-Ura-His/Leu的的）这些菌株。此外，冻结他们在25％甘油在-80℃下建议的附加试验，如野生型序列的情况下，删除的基因和脉冲场凝胶电泳确认。 </li><li>性循环重复（2.1），提供有用的菌株。 </li></ol>

<ol>
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Acids Res. 38, 2570-2576 (2010).</li><li>Suzuki, Y., 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=Knocking+out+multigene+redundancies+via+cycles+of+sexual+assortment+and+fluorescence+selection.">Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.</a> Nat. Methods. 8, 159-164 (2011).</li><li>Winzeler, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+of+the+S.+Genome+by+Gene+Deletion+and+Parallel+Analysis.">Functional Characterization of the S. Genome by Gene Deletion and Parallel Analysis.</a> Science. 285, 901-906 (1999).</li><li>Woods, R. A., Gietz, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+plasmid+DNA+into+yeast.">High-efficiency transformation of plasmid DNA into yeast.</a> Methods Mol. Biol. 177, 85-97 (2001).</li><li>Hughes, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+aneuploidy+revealed+by+DNA+microarray+expression+profiling.">Widespread aneuploidy revealed by DNA microarray expression profiling.</a> Nature. 25, 333-337 (2000).</li><li>Goldstein, A. L., McCusker, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+new+dominant+drug+resistance+cassettes+for+gene+disruption+in+Saccharomyces+cerevisiae.">Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.</a> Yeast. 15, 1541-1553 (1999).</li><li>Newman, J. R. S., 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=Single-cell+proteomic+analysis+of+S.+cerevisiae+reveals+the+architecture+of+biological+noise.">Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.</a> Nature. 441, 840-846 (2006).</li><li>Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+at+the+single-cell+level.">Gene regulation at the single-cell level.</a> Science. 307, 1962 (2005).</li></ol>

没有利益冲突的声明。

我们开发的绿色怪物的方法，我们关注的不同的GFP更换磁带的可能性之间的重组，从而导致基因组重排。成功地进行了多轮交配和减数分裂的细胞，减轻对这种可能性是我们的选择。细胞重新排列的基因组预计将不太适合交配后的单元格不相同的重排。事实上，我们并没有观察到任何导致基因组不稳定之间的重组GFP箱11。但是，我们不能完全排除这种可能性，所以建议用户测试生成的株重...

<table border="1"><tbody><tr><td> 的试剂或仪器的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> G418 </td><td> Sigma-Aldrich公司</td><td> A1720 </td><td>溶于水，过滤，消毒（0.2微米的过滤器）。股票浓度为200毫克/毫升。存放于4℃ </td></tr><tr><td> ClonNAT（nourseothricin） </td><td> WERNER BioAgents </td><td> 5001000 </td><td>溶于水，过滤，消毒（0.2微米的过滤器）。股票浓度为100毫克/毫升。存放于4℃ </td></tr><tr><td>多西环素</td><td> Sigma-Aldrich公司</td><td> D9891 </td><td>溶解在50％乙醇中，并过滤灭菌（0.2-μm过滤器）。股票浓度为10毫克/毫升。新鲜每四个星期。从光屏蔽用铝箔和存储在4°C。 </td></tr><tr><td> ZYMOLYASE </td><td> ZymoResearch </td><td> E1005 </td><td></td></tr><tr><td> Difco酵母氮基W / O型氨基酸</td><td> BD </td><td> 291940 </td><td></td></tr><tr><td>左轮手枪（旋转器管） </td><td> Labnet </td><td> H5600 </td><td></td></tr><tr><td>耐力赛凝胶XL电泳仪</td><td> Labnet </td><td> E0160 </td><td></td></tr><tr><td> SONIFIER 450 </td><td>布兰森</td><td> 101-063-198 </td><td></td></tr><tr><td>微尖SONIFIER 450 </td><td>布兰森</td><td> 101-148-062 </td><td></td></tr><tr><td> FACSAria细胞分选仪</td><td> BD </td><td></td><td></td></tr><tr><td> MoFlo细胞分选仪</td><td>贝克曼 - 库尔特</td><td></td><td></td></tr><tr><td> Biomek FX或相当于机器人</td><td> Beckman Coulter公司</td><td></td><td>可选。对于设立基因分型的PCR。 </td></tr></tbody> &lt;/ TABLE&gt;

the green monster process for the generation of yeast strains carrying multiple gene deletions

只有当测试是在一个特定的遗传背景或环境条件1,2的突变表型基因缺失往往发现。有这样的例子，许多基因需要被删除，揭露隐藏的功能基因3,4。尽管有潜在的重要发现，主要涉及三个或更多的基因的遗传相互作用的探索。多突变体相互作用的穷举搜索将是不切实际的因缺失可能的组合数量之多。然而，研究选定的几套基因，如套旁系同源具有更大的先验机会共享一个共同的功能，将信息。 酵母中的酿酒酵母（Saccharomyces cerevisiae）中 ，是通过更换通过同源重组的可选择标记基因与基因敲除。由于标记的数目是有限的，方法已被开发用于除去和重用的sam<html> Ë标记5,6,7,8,9,10。然而，依次工程多个突变，使用这些方法是耗时的，因为所需要的时间以产生缺失的数量呈线性变化关系。 在这里，我们描述了常规工程酵母11多个缺失的绿色怪物的方法。在该方法中，绿色荧光蛋白（GFP）报告用于定量标签菌株缺失纳入根据每种菌株中所含的缺失（ 图1）的数量。各种各样的GFP-标记的缺失通过酵母交配和减数分裂反复多轮耦合与流式细胞富集携带更多的这些缺失导致的积累缺失株（ 图2）的菌株。并行执行多个进程，与每个每轮将一个或多个缺失的方法，可以减少应变施工所需要的时间。 内容“&gt;的第一个步骤是，以制备单倍体单突变体被称为&#39;ProMonsters，其中每个执行在一个已删除的位点和”工具箱“位点之一的GFP记者要么绿色妖怪GMToolkit-a或GMToolkit-α在can1Δ位点（ 图3），使用从酵母缺失集合12株，GFP标记的缺失可以方便地产生取代共同KanMX4盒中存在的一个普遍GFP - URA3片段与这些菌株。包含每个GMToolkit：无论是一个 -或α-交配型特定单倍体选择标记1，准确的两个标记，当两个GMToolkits是本，统称允许二倍体选择之一。 第二个步骤是进行性循环通过的随机分类并/或减数分裂RECOMB的的缺失位点，通过它可以结合在一个单一的细胞ination，伴随着每一个周期的交配和产孢。

当携带4 GFP标记的缺失（ycl033cΔyer042wΔykl069wΔyol118cΔ） 的 a-单倍体菌株杂交的α-单倍体菌株携带四个缺失（ycl033cΔydl242wΔydl227cΔyer042wΔ），有两个缺失（ycl033cΔyer042wΔ）共享由两个菌株，代表结果得到的。培养的YPDA培养基中G418和NAT选择二倍体交配混合物。将得到的二倍体在孢子形成的介质中培养。将孢子分散治疗和超声ZYMOLYASE，，发芽在单倍体选择媒体。然后将...

Watch this Scientific Journal Video about The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions at JoVE.com

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

绿色妖怪方法能够快速组装的多个标记的报道基因编码绿色荧光蛋白的缺失。这种方法是基于对驾驶酵母菌株通过各种各样的缺失性和携带更多的缺失的细胞的荧光为基础的浓缩的重复循环。

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

the-green-monster-process-for-generation-yeast-strains-carrying

Research

JoVE Journal

Biology

14.2K 次观看.  J. Craig Venter Institute. 绿色妖怪方法能够快速组装的多个标记的报道基因编码绿色荧光蛋白的缺失。这种方法是基于对驾驶酵母菌株通过各种各样的缺失性和携带更多的缺失的细胞的荧光为基础的浓缩的重复循环。

视频: The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...