这项工作是支持的补助金从美国国立卫生（PX）和R01DE018138部分，弗吉尼亚联邦大学校长科研奖励计划（PRIP）144602-3（PX）。我们感谢博士。雷震，西城区月坛窦和小静王为协助建设的全基因组突变体。我们也感谢的DNA核心设施在弗吉尼亚联邦大学进行DNA测序。

1。引物设计<ol><li>引物的设计采用了内部脚本的基础上的S.血统的 SK36基因组序列。三套引物，F1/R1，F2/R2和F3/R3被设计用于扩增的1-kb的上游序列的靶基因，APHA-3基因编码卡那霉素抗性（公里R）蛋白3和1 -kb的下游序列的靶基因，分别（ 图1）。在这些引物中，F1和R 3的设计采用ePrimer3 EMBOSS套件的方案（ <a href="http://emboss.sourceforge.net/apps/cvs/emboss/apps/index.html" target="_blank">http://emboss.sourceforge.net/apps/cvs/emboss/apps/index.html</a> ）扩增侧翼区的上游或下游的目标基因。基因特异性引物，R 1和F3，设计根据的5&#39;和3&#39;序列的靶基因。 R1和F3引物包含25 bp的适配器，在其5&#39;末端的序列是互补的APHA-3的基因。各引物的熔化温度被设计为尽可能接近到60℃，以使均匀的退火温度可用于所有PCR反应在96孔格式的使用。 </li><li> F1，F3，R1和R3引物合成的基因顺序的基础上在96 - 孔板中。每块板包含一种类型的引物在相同的基因顺序。稀释在96 - 孔工作引物板的引物进行PCR扩增，使用多道移液器中至最终浓度为10μM。 </li></ol> 2。高通量PCR扩增，纯化<ol><li>以高通量扩增出1 kb的上游或下游的目标S.血统基因，组装的PCR鸡尾酒混合物在冰中含有1640微升DDH 2 O，250微升10xHigh富达PCR缓冲液，200微升10mM的dNTP混合液，加入100μl的50 mM MgSO 4干燥 ，加入100μl的15毫升锥形管10 ng /μl的S。血统 SK36的基因组DNA和10μl的Taq DNA铂polymeRASE高的保真度和传输23微升的混合物中，每孔96孔PCR板，使用多道移液器。 1微升每个F1和R1（或F3和R3）的传输工作引物板的PCR板使用多道移液器从10μM。密封PCR板和执行放大，在94℃1分钟，30个循环的94℃30秒，54℃下为30秒和68℃1.5分钟。 </li><li>准备1％琼脂糖凝胶含有溴化乙锭的48孔嵌合多道移液器装载。 4微升的PCR产物用1微升5xDNA样缓冲液混合，在96孔板上，并加载在琼脂糖凝胶上的样品，使用10微升的多道移液器。运行在135 V电泳30分钟。检查带凝胶，UVP记录和分析系统。 </li><li>纯化的PCR产物由96 PureLink PCR纯化试剂盒，根据制造商的指示的使用离心。为了洗脱DNA，加入40μl的无菌DDH 2 O的B井inding板。 </li><li>随机挑选几个纯化扩增板使用的NanoDrop分光光度计检查的DNA浓度。铭牌上的扩增子的浓度调节到〜10纳克/微升。 </li><li>消化的质粒公里ŕ磁带使用ECOR我作为PCR模板。消化的质粒通过QIAquick PCR纯化试剂盒纯化和线性质粒DNA被调整到最终DNA浓度10纳克/微升。组装25微升混合物公里ŕ盒扩增子包括16.4微升DDH 2 O，2.5微升10xHigh富达PCR缓冲液，2微升10mM的dNTP混合液，1微升的50mM的Mg SO 4，10μMF2，1微升1微升10μM的R2，1微升的线性质粒DNA和0.1微升白金的Taq DNA聚合酶的高保真度。进行PCR扩增，在94℃1分钟，30个循环的94℃30秒，55℃下为30秒和68℃下进行1分钟。检查的PCR产物进行1％琼脂糖凝胶电泳。蒲慕明洛杉矶大部分的Kmŕ由10个独立的PCR纯化QIAquick PCR纯化试剂盒磁带扩增子。调整的扩增子浓度为10 ng /μL。 </li><li>以得到最终的线性重组PCR扩增子，3个PCR扩增子相结合，与每1微升（几乎相等的摩尔量），在一个PCR板以及作为PCR模板。其他PCR成分被添加，包括14.4微升DDH 2 O，2.5微升10xHigh富达PCR缓冲液，2微升10mM的dNTP混合液，1微升的50mM的Mg SO 4，10μM的F1，1微升1微升10μM的R3， 0.1μL铂金的Taq DNA聚合酶高的保真度。 PCR扩增在94℃下进行30秒55℃2分钟，30个循环的94℃下为30秒和68℃3.5分钟，最后68℃4分钟。 </li><li>检查PCR产物在琼脂糖凝胶上。如上所述净化和量化的PCR扩增子。冻结重组PCR扩增产物在-20°C。 </li></ol> 3。 COMpetent的制备<ol><li> Todd Hewitt肉汤制备，调节pH值至7.6，用10 1N的NaOH，加热至沸腾，然后冷却至室温，并灭菌过滤器4使用0.22微米聚苯乙烯。加入300μl的热灭活马血清（终浓度为2.5％）至11.7毫升Todd Hewitt肉汤15毫升锥形管，以弥补TH + HS介质。等分试样TH + HS介质到2毫升和10毫升管中。 </li><li>接种5μL的股票S.血统 SK36到2ml TH + HS介质和在-80℃下冻结，孵育培养过夜封端紧紧地在37℃下，伴随着预孵育10毫升TH + HS管。 </li><li>过夜后，转移到10ml TH + HS50μl的文化，管在37℃下孵育3小时（对应0.07-0.08的OD 660），立即使用用于转化。 </li></ol> 4。细胞转化和抗生素的选择<ol><li>加入2μl70毫微克S.血统 SK36 COMPE唐塞刺激肽（CSP）和2微升线性重组PCR扩增子（〜50毫微克）到Eppendorf管中，在96孔块和预暖它们在37℃下每管330μl的3小时孵育SK36文化的转移。在37℃下孵育1小时。替换DNA，用无菌DDH 2 O控制。 </li><li>块放置在冰上，并与500微克/毫升卡那霉素的脑心浸液（BHI）琼脂平板上传播100μl的每个变换。在37°C孵育板微氧条件下为2天。 </li></ol> 5。突变的确认和存储<ol><li>每次更换突变体，随机挑选2个单个菌落，接种到5毫升BHI为500μg/ ml卡那霉素的殖民地和孵化接种microaerobically隔夜在37°C。 30％的甘油冷冻保存每一种文化在-80°C </li><li>要检查是否包含预期的基因替代突变体，每一个突变F1进行菌落PCR和R3引物在96孔PCR板。约1微升过夜培养从单个菌落作为模板DNA的25微升的PCR扩增反应中使用。进行PCR，在94℃5分钟，35个循环的94℃下30秒，55℃3.5分钟，最后68℃下4分钟为30秒和68℃下进行。 </li><li>为了精确地确定双波段的突变体或污染物的PCR扩增，通过电泳检查PCR扩增子，在&gt; 12厘米长的2％琼脂糖凝胶用溴化乙锭染色的4小时。这琼脂糖凝胶电泳条件下，任何扩增产物≥100 bp的差异进行明确标识。当频带从公里ŕ盒扩增的野生型基因所产生的预期&lt;100，一个内部的T1引物使用，以确定是否可以通过PCR检测的野生型基因的沸点不同。 </li><li>为了进一步确认删除时，PureLink 96 PCR纯化试剂盒纯化，扩增子的使用P1引物和测序结合公里ŕ盒。只保留正确的突变体经测序证实。 </li></ol>

<ol>
	<li>Sassetti, C. M., Boyd, D. H., Rubin, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+mycobacterial+growth+defined+by+high+density+mutagenesis.">Genes required for mycobacterial growth defined by high density mutagenesis.</a> Mol. Microbiol. 48, 77-84 (2003).</li><li>Kobayashi, K., 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=Essential+Bacillus+subtilis+genes.">Essential Bacillus subtilis genes.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4678-4683 (2003).</li><li>Trieu-Cuot, P., Courvalin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+the+Streptococcus+faecalis+plasmid+gene+encoding+the+3'5"-aminoglycoside+phosphotransferase+type+III.">Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III.</a> Gene. 23, 331-341 (1983).</li><li>Paik, 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=Identification+of+virulence+determinants+for+endocarditis+in+Streptococcus+sanguinis+by+signature-tagged+mutagenesis.">Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.</a> Infect. Immun. 73, 6064-6074 (2005).</li><li>Lukomski, 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=Nonpolar+inactivation+of+the+hypervariable+streptococcal+inhibitor+of+complement+gene+(sic)+in+serotype+M1+Streptococcus+pyogenes+significantly+decreases+mouse+mucosal+colonization.">Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization.</a> Infect. Immun. 68, 535-542 (2000).</li><li>Baba, T., 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=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Mol. Syst. Biol. 2, (2006).</li><li>de Berardinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complete+collection+of+single-gene+deletion+mutants+of+Acinetobacter+baylyi+ADP1.">A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.</a> Mol. Syst. Biol. 4, 174 (2008).</li><li>Rodriguez, A. M., 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=Physiological+and+molecular+characterization+of+genetic+competence+in+Streptococcus+sanguinis.">Physiological and molecular characterization of genetic competence in Streptococcus sanguinis.</a> Mol. Oral. Microbiol. 26, 99-116 (2011).</li><li>Perry, D., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Streptococcus+mutans+Infect.">Genetic transformation of Streptococcus mutans Infect.</a> Immun. 32, 1295-1297 (1981).</li><li>Pakula, R., Walczak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+of+competence+of+transformable+streptococci.">On the nature of competence of transformable streptococci.</a> J. Gen. Microbiol. 31, 125-133 (1963).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Xu, P., 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=Genome-wide+essential+gene+identification+in+Streptococcus+sanguinis.">Genome-wide essential gene identification in Streptococcus sanguinis.</a> Sci. Rep. 1, (2011).</li></ol>

没有利益冲突的声明。

为了尽量减少可能的极性的影响1与外源性抗生素基因对邻近基因的靶基因的更换，采取两个步骤，而最初的引物设计。对于大多数已删除的基因，引物R1和F3被设计用于删除后的6个碱基的30 bp的起始密码子到终止密码子之前的编码区。最后保留了30个碱基对，以保护潜在的使用相邻的下游基因的核糖体结合位点。两个相邻的基因之间的保留区域被扩展到100 bp的，当他们位于相反方向，这两种蛋白的...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;姓名</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品编号</strong</td&gt;<td<strong评论（可选）</strong</td&gt;</tr&gt;<tr&gt;<td&gt;引物F2</td&gt;<td集成DNA技术</td&gt;<td</td&gt;<td&gt; TGACTAACTAGGAGGAATAAATG<br/&gt; GCTAAAATGAGAATAT</td&gt;</tr&gt;<tr&gt;<td&gt;入门R2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CATTATTCCCTCCAGGTACTAAAA<br/&gt; CAATTCATCCAGT</td&gt;</tr&gt;<tr&gt;<td&gt;入门F2P</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GATAAACCCAGCGAACCATTTGA</td&gt;</tr&gt;<tr&gt;<td&gt;引物P1</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCTTATATACCTTAGCAGGAGACA</td&gt;</tr&gt;<tr&gt;<td&gt;引物P2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTATGACATTGCCTTCTGCGTCC</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_R1_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCCATTTATTCCTCCTAGTTAGTCA</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_F3_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTTTTAGTACCTGGAGGGAATAATG</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_R1p_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CCTCAAATGGTTCGCTGGGTTTATC</td&gt;</tr&gt;<tr&gt;<td&gt; CSP</td&gt;<td&gt; Synprep</td&gt;<td</td&gt;<td&gt;序列：NH 2-DLRGVPNPWGWIFGR-COOH的</td&gt;</tr&gt;<tr&gt;<td&gt; DNA聚合酶</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 11304-102</td&gt;<td&gt;铂金Taq DNA聚合酶高保真</td&gt;</tr&gt;<tr&gt;<td&gt; EcoRI位</td&gt;<td&gt;新英格兰生物实验室公司</td&gt;<td&gt; R0101S</td&gt;<td&gt;限制性内切酶</td&gt;</tr&gt;<tr&gt;<td&gt;琼脂</td&gt;<td美国生物分析</td&gt;<td&gt; AB01185</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;琼脂糖</td&gt;<td&gt; Promega公司</td&gt;<td&gt; V3125</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; BHI</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 237500</td&gt;<td&gt;脑心浸液</td&gt;</tr&gt;<tr&gt;<td&gt;马血清</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; SH3007403</td&gt;<td&gt;马血清</td&gt;</tr&gt;<tr&gt;<td&gt; KM</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; BP906-5</td&gt;<td&gt;卡那霉素</td&gt;</tr&gt;<tr&gt;<td&gt; TH肉汤</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 249240</td&gt;<td&gt; Todd Hewitt肉汤</td&gt;</tr&gt;<tr&gt;<td&gt; PureLink 96</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; K310096</td&gt;<td&gt; PureLink 96 PCR纯化试剂盒</td&gt;</tr&gt;<tr&gt;<td&gt; QIAQUICK</td&gt;<td&gt; Qiagen公司</td&gt;<td&gt; 28106</td&gt;<td&gt;的Qiaquick PCR产物纯化试剂盒</td&gt;</tr&gt;<tr&gt;<td&gt;过滤器</td&gt;<td&gt;康宁</td&gt;<td&gt; 431117</td&gt;<td&gt; 0.22微米的聚苯乙烯过滤器</td&gt;</tr&gt;<tr&gt;<td&gt; Anoxomat系统</td&gt;<td&gt;沃尔玛微生物BV</td&gt;<td&gt; Anoxomat Mark II的</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;台式离心机</td&gt;<td&gt;赛默飞世尔科技</td&gt;<td&gt; 75004377</td&gt;<tdSORVALL传奇RT Plus微孔板转子的</td&gt;</tr&gt;<tr&gt;<td&gt;凝胶</td&gt;<td&gt; UVP有限责任公司</td&gt;<td&gt; BioDoc-210</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;孵化器</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; 11-690-625D</td&gt;<td&gt; Isotemp</td&gt;</tr&gt;<tr&gt;<tdLabnet的凝胶XL</td&gt;<td&gt; Labnet</td&gt;<td&gt; E0160</td&gt;<tdLabnet的凝胶XL</td&gt;</tr&gt;<tr&gt;<td&gt;微型离心机</td&gt;<td&gt; Eppendorf公司</td&gt;<td&gt; 22621408</td&gt;<td&gt;微量5415ŕ</td&gt;</tr&gt;<tr&gt;<td&gt;多道移液器</td&gt;<td&gt;热Scientfic</td&gt;<td&gt; 4661040，4661020</td&gt;<td&gt; Finnpipette F1</td&gt;</tr&gt;<tr&gt;<td&gt;热循环仪</td&gt;<td&gt;应用生物系统公司</td&gt;<td&gt; N805-0200</td&gt;<td&gt; GeneAmp PCR系统9700</td&gt;</tr&gt;</tbody&gt;</table

genome-wide gene deletions in streptococcus sanguinis by high throughput pcr

转座子突变和单基因缺失的两种方法适用于全基因组基因敲除细菌1,2。虽然转座子诱变是耗时更少，成本更低，并且不需要完成的基因组信息，在该方法中有两个缺点：（1）在该混合反选择竞争降低的突变体的突变体库的一个完全不同的突变体的可能性;和（2）的部分的基因失活的可能性，即基因的不完全失去的转座子的插入后，它们的功能。单基因的缺失分析可能补偿相关的缺点与转座子诱变。为了提高效率的全基因组的单基因缺失，我们尝试建立了高通量技术的使用链球菌血统作为模式生物的全基因组单基因缺失。每个基因缺失构建S.血统基因组被设计成包括1-kb的上游的靶基因，APHA-3基因，编码卡那霉素抗性的蛋白质，和1-kb的下游的靶基因。分别三套引物F1/R1 F2/R2和F3/R3，设计并合成用于PCR扩增这三个组成部分的每个删除构建在96孔板中的格式。引物R1和F3包含25-bp的序列的APHA-3基因在它们的5&#39;末端的区域是互补的。执行一次创建所有的单基因缺失构建一个大型的APHA-3基因的 PCR扩增。 APHA-3基因的启动子的初步排除卡那霉素磁带，以减少潜在的极性效应。要创建的基因缺失构建，高吞吐量的PCR扩增和纯化是在96孔板中的格式进行。线性每个基因缺失的重组PCR扩增子将通过4采用高保真DNA聚合酶的PCR反应。最初的expon差分生长阶段的S.血统 Todd Hewitt肉汤培养在补充有2.5％灭活的马血清是用来增加PCR的重组构建体的转化能力。在此条件下，高达20％的S.血统细胞可以使用〜50ng的DNA转化。根据此方法，2048个单基因缺失的突变体，最终获得的2270个基因在S.血统不包括4个基因的ORF完全包含在其他的ORF S.血统 SK36和218潜在的必需基因。该技术是高通量基因缺失结构，可以很容易使用任何变形菌的全基因组的单基因缺失。

经过PCR扩增用引物F1和R1，和F3和R3，约1 kb的上游和下游的每个S.血统基因中得到的96孔板的形式，分别为（ 图2A）。在我们的PCR条件下，使用设计引物，扩增特定产品的S.血统每个PCR反应中的基因组DNA。此结果表明，该引物对具有高度特异性的目标的S.血统 。通过PCR扩增使用F1和R3引物和3扩增子作为DNA模板，线性的重组PCR构造（〜3 kb的）的高通量的96孔板中，组成的...

Watch this Scientific Journal Video about Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR at JoVE.com

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

已经建立一个高效的全基因组单基因突变的方法使用<em&gt;链球菌血统</em作为一种模式生物。通过这种方法已经取得了高通量重组PCR和转换。

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 ...

genome-wide-gene-deletions-streptococcus-sanguinis-high-throughput

Research

JoVE Journal

Biology

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

16.4K 次观看.  Virginia Commonwealth University. 已经建立一个高效的全基因组单基因突变的方法使用链球菌血统

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

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

科研

生物学

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

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. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...