pMSCV - AID - I - Thy1.1和pKat2载体，从DG Schatz和pVSV -摹，PRSV修订版的礼物，并pMDLg / pRRE载体是一种从BR脐周礼物。

1。准备样品和细胞<ol><li>执行一个中等规模制备的DNA（midiprep）的过度表达或shRNA包含结构和逆转录病毒包装载体pKat2或慢病毒包装载体pVSV - G，pMDLg / pRRE和PRSV - 冯 25 。使用每个构造中的DNA和6微克4微克pKat2（逆转录病毒感染）或每个pVSV - G，pMDLg / pRRE 1.5微克DNA，PRSV - REV包装载体（慢病毒感染）。 </li><li>准备BOSC 23家媒体。转染，使用两个未经修正的贝科的改良Eagle培养基（DMEM），以及完全DMEM。为了使完全DMEM，添加1％HEPES缓冲液，1％青霉素，链霉素，谷氨酰胺（PGS），和10％新生牛血清（NCS）一瓶的DMEM。 </li><li>准备拉莫斯媒体。对于感染的步骤，完成一瓶的RPMI加入的1％HEPES，1％的PGS，10％胎牛血清（FBS），RPMI - 1640培养基（RPMI）。对于单细胞播种步骤，MAKE"空调"的媒体拉莫斯细胞生长在完全RPMI 2天，纺纱400 X克细胞4分钟，过滤取上清液用0.2微米的过滤器进入无菌瓶。其他完整的媒体一样，条件培养基保存于4 ° C直到需要。 </li><li>拆分3 × 10 6 BOSC 23到100毫米的钢板在10毫升完全DMEM细胞，细胞转染前一天，将在50-60％汇合，转天。使每个向量计划转染细胞板。重要的是时间过长，这些细胞没有被保存在文化;使用低通过数量最近解冻的细胞或细胞转染和感染的措施，以确保良好的感染。 </li><li>包括控制你的实验。对于转染和感染，创建一个阴性对照（未转染或未受感染的细胞）和阳性对照（病毒表达GFP和嘌呤霉素抗性基因）。 shRNA的敲下来experiments，感染与杂乱或无关的shRNA控制良好。 </li></ol> 2。 BOSC转染细胞23 <ol><li>未经修正的DMEM和完全DMEM瓶温在37 ° C。 </li><li> BOSC 23细胞中删除的媒体，并与5毫升完全DMEM取代。 37 ° C培养箱，直到返回的细胞在步骤2.6中使用。 </li><li>暖FuGENE 6瓶在室温下5分钟，然后简要涡（&lt;1秒）。 </li><li> 1.5 ml离心管分装成600μL，未经修订的DMEM。 </li><li>未经修订的DMEM 600μL，涡旋简要（&lt;1秒）稀释成30μL，FuGENE 6，然后在室温下孵育五分钟。小心吸管直接进入媒体。不要让FuGENE 6触摸管双方。 </li><li> 6微克构造，无论是4微克pKat2（逆转录病毒感染）或1.5微克每pVSV - G pMDLg / pRRE，PRSV - REV（慢病毒感染）的DMEM / FuGENE 6混合，涡旋BRI<html> EFLY（&lt;1秒），然后在室温下孵育25分钟。 </li><li>添加BOSC 23细胞的DNA / DMEM / FuGENE 6混合和回报为24小时37 ° C培养箱。 </li><li> 24小时后，加入5毫升完全DMEM转染细胞和回报为24小时，37 ° C培养箱。 </li></ol> 3。收获的病毒颗粒<ol><li>收获转BOSC 23细胞用10 mL注射器媒体10毫升。 </li><li>附加0.45μm的过滤器，注射器和过滤到一个干净的15 mL聚丙烯管的媒体。 </li><li>立即使用病毒的媒体为1毫升分装在2 mL低温瓶或冻结在-80 ° C。 </li><li>收获后的病毒媒体，确认，高效的转染阳性对照细胞发生流式细胞仪。这也可以为所有样品，如果结构包含一个荧光（如GFP）或细胞表面标志物（如Thy1）。 </li></ol>书名"&gt; 4。感染B细胞<ol><li>种子2毫升2 × 10 5细胞/毫升完全RPMI拉莫斯细胞到感染前24小时，6孔板。再次，一定要使用低传代次数最近解冻的细胞或细胞，以确保良好的感染。 </li><li>如果使用冷冻的病毒等份，病毒的媒体迅速解冻在37 ° C。 </li><li>混合3μL聚凝胺10毫克/毫升1毫升病毒媒体。对聚凝胺终浓度应总量的3毫升10微克/毫升。 </li><li>细胞和病毒/聚凝胺混合拌匀吹打。如前所述较早，以及应被用来作为一个未受感染的控制;加入1 mL完全RPMI和3μL，聚凝胺这口井，而不是1毫升病毒。请务必感染匹配的阴性对照。我们也传染以及含有GFP的载体，流式细胞仪，以确定感染效率。 </li><li>在室温为90分钟，离心3000 X克在6孔板。 </li><li>返回板块37℃培养箱中培养48小时。 </li><li> 48小时后，400 X克降速感染的细胞在室温下4分钟在15毫升的聚丙烯管。吸取出媒体，确保不扰乱细胞沉淀。在新鲜的RPMI重悬细胞。 </li><li>确认感染流式细胞仪阳性对照样品的效率。像以前一样，可以为您的样品，如果结构包含一个荧光（如GFP）或细胞表面标志物（如Thy1）。 </li><li>开始选择的单元格区域（如适用）。我们的shRNA包含结构中都含有嘌呤霉素抗性卡带。我们选择shRNA的感染拉莫斯细胞，用0.25μg/ mL的嘌呤。我们发现，拉莫斯细胞的衍生工具，有时显示改变到嘌呤霉素的敏感性，因此，我们执行杀以优化每个衍生嘌呤浓度的曲线。也可治疗嘌呤霉素未受感染的控制细胞，以确认选择是有效的的。 </li></ol> &lt;P类="j​​ove_title"&gt; 5。单细胞种植 单播可以在以下两种方式之一进行:排序或手动流式细胞仪。 <ol><li>流式细胞仪:一个流式细胞仪分选机板适配器使用种子1细胞/孔在96孔板100μL40％RPMI/40％，空调media/20％FBS预填充。此方法提供了很多更多的单细胞克隆，每盘。 </li><li>手动的细胞计数。 RPMI/40％空调media/20％FBS的40％稀释到10个细胞/毫升的细胞等分。使用10毫升稀释的细胞为每个种子板块。不同的克隆将显示在这个阶段变量恢复，因此它是有用的设立在不同浓度（从3到20个细胞/毫升）的几个板块。使用产生较少的副产物，以避免从多个细胞产生的克隆）。使用多道移液器种子96孔板100μL稀释细胞。 </li><li>放置在37℃培养箱中培养2周的板块。 </li><li>检查个别井undeR的显微镜确认细胞的存在一天后播种。单细胞可以比较难找到，由于需要集中在正确的深度。聚焦在96孔板的底部印以及数字应该有所帮助。细胞很容易发现，因为多数井有细胞流式细胞仪分选机种子。 </li><li> 2周后，越来越多的殖民地，可以勉强看到肉眼看从下面板块。他们要容易得多，在显微镜下看到。殖民地往往一起成长的好，通常在平板的菌落生长在不同的井边缘的边缘。马克井具有强大的细胞生长和转移的细胞在1 mL的RPMI的24孔板，并返回到37 ° C培养箱。 </li><li>保持在1 × 10 5至1 × 10 6细胞/ mL的细胞。更改为4分钟，在室温下旋转400 X克的细胞和消除媒体的媒体。更换用1 mL新鲜的RPMI。 </li><li>经过几个ðays的增长，细胞转移到6孔板，在37℃培养箱继续增长。 </li><li> 3周后，分析IgM的损失（如下所述）的克隆。 </li><li>继续增长2个星期的细胞，然后分析，在5周时间点的克隆。 </li></ol> 6。分析:IgM的损失（3周和5周） <ol><li>分装5 × 10 5细胞，从每口井到1.5 ml离心管和旋转400 X克细胞在室温为4分钟。使用未染色控制细胞的控制和击倒的shRNA井，以及一个样本。 </li><li> 1毫升的PBS洗涤细胞，然后再旋转400 X克在室温为4分钟。 </li><li>在50μL的PBS（未染色控制）或PBS悬浮+ 1:20稀释的α-人IgM抗体PE标记。在冰上孵育1小时。 </li><li>在室温下4分钟旋转400 X克细胞。 </li><li>洗的C英语学习者的两倍，1毫升的PBS和自旋为4分钟，每一次都在室温下为400 X克。 </li><li>删除上清100μL的PBS和悬浮。悬浮染细胞转移一个流式细胞仪管。 </li><li>分析细胞，使用流式细胞仪机。 </li><li> IgM的百分比-从与shRNA的拆装下来的细胞的控制细胞进行比较。如前所述，流式细胞仪测定IgM的损失是一个可靠的半定量拉莫斯细胞SHM措施。 </li></ol> 7。分析:确认击倒定量RT - PCR（5周） <ol><li>分装3 × 10 6细胞，从每口井和旋转400 X克在室温为4分钟。 </li><li>准备等分细胞的RNA。我们使用TRIZOL根据制造商的建议。 </li><li>准备从基因的RNA的准备。我们使用的是2微克的RNA和上标II根据制造商的建议。 </li><li>执行定量PCR使用基因confir米敲下来。我们作为一个正常化的控制，并在10μL的反应总体积为SYBR FAST的qPCR试剂盒GAPDH的。 </li></ol> 8。分析:基因组测序（5周） <ol><li>分装5 × 10 6细胞，从每口井和旋转400 X克在室温为4分钟。 </li><li>隔离这些细胞的基因组DNA。我们使用向导SV基因组DNA纯化试剂盒，根据制造商的建议。 </li><li> PCR扩增的Ig V基因19（见表1），使用KAPA高保真DNA聚合酶的利益的任何其他基因的基因组DNA。我们利用聚合酶，因为其低误码率，但任何高保真PCR聚合酶应该是可以接受的的。 </li><li>凝胶纯化PCR产物在1％琼脂糖凝胶使用QIAquick凝胶回收试剂盒。 </li><li>有些聚合酶添加一个单一的脱氧（一）残留物的PCR产物的3&#39;末端，而有的则没有。这3个"A尾巴是必要的TOPO TA克隆的一步。 KAPA高保真聚合酶不留3&#39;一个尾巴。一尾孵化与25μL凝胶纯化PCR产物3μL，10倍的Taq反应缓冲液，1μL10 mM的dNTPs，并在72℃30分钟1单位Taq聚合酶的PCR产物。 </li><li>克隆凝胶纯化的PCR产物使用TOPO TA克隆试剂盒。 TOPO的反应转化到大肠杆菌的产品大肠杆菌 DH5α- T1 ř能干细胞和含有100μg/ mL氨苄青霉素，并根据制造商的建议，补充1.6毫克X - gal的LB /琼脂板的板。 </li><li>发送单个菌落进行测序。 </li><li>执行序列分析，以确定突变的类型和位置。 </li></ol> 9。代表性的成果: 如前所述，我们使用阳性对照病毒载体表达GFP，以及嘌呤霉素抗性基因。我们通常看到的50-75％GFP +细胞，两天转染后。在我们的感染，我们通常看到的50-70％的细胞 +两天后感染，但前选择绿色荧光蛋白。选择完成后，&gt; 95％的细胞GFP +。 为代表的实验中，我们展示过表达的援助或撞倒拉莫斯细胞修复因子的影响。具体来说，我们转了援助逆转录病毒的过度表达载体，通过IRES的网站一起，与逆转录病毒包装载体pKat2 Thy1.1 BOSC 23细胞相连。含有病毒的媒体过滤，然后用来感染拉莫斯细胞。野生型（WT）和援助过表达细胞（援助您好 ）单细胞播种和生长3个星期，在这一点，他们的基因表达分析QRT - PCR（图2）和流式细胞仪IgM的损失（图3 ）。流式细胞仪染色，细胞培养与两个1:20稀释PE标记的α-人IgM抗体以及1:400稀释FITC标记的α-大鼠CD90/mouseCD90.1（Thy1.1）抗体。在另一项实验中中，包含无关的shRNA（对照组）或对高保真的DNA修复，预计防止SHM因素的shRNA慢病毒BOSC 23细胞，然后感染到拉莫斯细胞克隆援助您好。同样，基因表达（图4）和IgM的损失（图5）进行了分析，单细胞克隆后3个星期。由于修复因子被认为是提供对保护在SHM的突变，我们看到在IgM的损失和SHM因素的情况下增加。如果我们下来撞倒在参与生成SHM - 相关突变的一个因素，我们将看到在IgM的损失减少。 正如预期的那样，我们的QRT - PCR结果显示，援助表达援助的过度表达载体（图2）感染细胞后显着增加，而针对性的shRNA对这一因素的存在DNA修复因子下降的水平（图4 ）。因为个人酷龙ES可能会有所不同，你可能会看到一些在基因表达水平的变化。因为基因的表达可以变化，有必要确认您计划进一步分析每个克隆表达。对同一基因的不同的shRNA可能有不同的基因表达的影响一样，所以你应该屏幕几个的shRNA，以确定其中影响最大的。也可能被击倒在蛋白质水平证实，免疫印迹。同样，单个克隆IgM的损失程度可能差别很大。一些克隆可能会表现出"中奖"的效果，IgM的突变发生的最早的细胞分裂之一-例如，你会看到&gt; 50％的IgM损失，仅仅是因为细胞成为IgM抗体-在两细胞阶段。这些克隆的影响最小化的几个单细胞克隆的中位数计算。 <img alt="图1" src="/files/ftp_upload/3573/3573fig1.jpg" /> 跨示意图如图1。染，感染和分析IgM的损失。有关详细信息，请参阅文本。 <img alt="图2" src="/files/ftp_upload/3573/3573fig2.jpg" /> 图2。拉莫斯细胞的援助表达。 WT和援助您好拉莫斯细胞单细胞克隆和生长3个星期，在这点在单个克隆援助表达QRT - PCR测定。 GAPDH的表达被用于正常化。 WT设置为1。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图3" src="/files/ftp_upload/3573/3573fig3.jpg" /> 图3。增加援助您好细胞IgM的损失相比，野生型细胞。在3个星期的时间点的流式细胞仪，表面IgM测定WT 和援助您好细胞IgM的损失的百分比计算。 （一）代表流式细胞仪积了WT克隆和援助您好克隆。的援助过表达Vector已通过IRES的站点链接到Thy1.1的援助，所以Thy1.1表达是用来作为援助表达的替代品。 （二）定量几个WT和援助您好克隆IgM的损失。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图4" src="/files/ftp_upload/3573/3573fig4.jpg" /> 图4。击倒在援助您好细胞修复因子。一个不相干的shRNA控制（控制）或我们的利益的shRNA（shRNA）的，和单细胞种子细胞感染。 3周后，在个别克隆基因表达的QRT - PCR测定。 GAPDH的表达被用于正常化。 WT设置为1。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图5" src="/files/ftp_upload/3573/3573fig5.jpg" /> 图5增加IgM的损失。拆装修复因子水平的援助您好细胞。流式细胞仪在3个星期的时间点，在几个克隆的表面IgM的测定和IgM的损失的百分比计算细胞仍然过度表达的援助。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img src="/files/ftp_upload/3573/3573table1.jpg" alt="表1" /> 表1:19的Ig基因引物序列:恒定区Cμ，重链可变区（V H），和轻链可变区（V L） 。

<ol>
	<li>Di Noia, J. M., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibody+somatic+hypermutation.">Molecular mechanisms of antibody somatic hypermutation.</a> Annu. Rev. Biochem. 76, 1-22 (2007).</li><li>Peled, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+somatic+hypermutation.">The biochemistry of somatic hypermutation.</a> Annu. Rev. Immunol. 26, 481-511 (2008).</li><li>Longerich, S., Basu, U., Alt, F., Storb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+in+somatic+hypermutation+and+class+switch+recombination.">AID in somatic hypermutation and class switch recombination.</a> Curr. Opin. Immunol. 18, 164-174 (2006).</li><li>Bardwell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+somatic+hypermutation+and+reduced+class-switch+recombination+in+exonuclease+1-mutant+mice.">Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.</a> Nat. Immunol. 5, 224-229 (2004).</li><li>Martomo, S. A., Yang, W. W., Gearhart, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Msh6+but+not+Msh3+in+somatic+hypermutation+and+class+switch+recombination.">A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.</a> J. Exp. Med. 200, 61-68 (2004).</li><li>Phung, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypermutation+at+G+and+C+nucleotides+in+immunoglobulin+variable+genes+from+mice+deficient+in+the+MSH2+mismatch+repair+protein.">Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.</a> J. Exp. Med. 187, 1745-1751 (1998).</li><li>Rada, C., Ehrenstein, M. R., Neuberger, M. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spot+focusing+of+somatic+hypermutation+in+MSH2-deficient+mice+suggests+two+stages+of+mutational+targeting.">Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.</a> Immunity. 9, 135-141 (1998).</li><li>Rada, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+isotype+switching+is+inhibited+and+somatic+hypermutation+perturbed+in+UNG-deficient+mice.">Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.</a> Curr. Biol. 12, 1748-1755 (2002).</li><li>Delbos, F., Aoufouchi, S., Faili, A., Weill, J. C., Reynaud, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+the+sole+contributor+of+A/T+modifications+during+immunoglobulin+gene+hypermutation+in+the+mouse.">DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.</a> J. Exp. Med. 204, 17-23 (2007).</li><li>Masuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+a+limiting+factor+for+A:T+mutations+in+Ig+genes+and+contributes+to+antibody+affinity+maturation.">DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.</a> Eur. J. Immunol. 38, 2796-2805 (2008).</li><li>Lim, D. S., Hasty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+mouse+rad51+results+in+an+early+embryonic+lethal+that+is+suppressed+by+a+mutation+in+p53.">A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.</a> Mol. Cell. Biol. 16, 7133-7143 (1996).</li><li>Xiao, Y., Weaver, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+targeted+deletion+by+Cre+recombinase+demonstrates+the+requirement+for+the+double-strand+break+repair+Mre11+protein+in+murine+embryonic+stem+cells.">Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.</a> Nucleic. Acids. Res. 25, 2985-2991 (1997).</li><li>Zhu, J., Petersen, S., Tessarollo, L., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+Nijmegen+breakage+syndrome+gene+NBS1+leads+to+early+embryonic+lethality+in+mice.">Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.</a> Curr. Biol. 11, 105-109 (2001).</li><li>Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mRad50+causes+embryonic+stem+cell+lethality,+abnormal+embryonic+development,+and+sensitivity+to+ionizing+radiation.">Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 7376-7381 (1999).</li><li>Pavri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+cytidine+deaminase+targets+DNA+at+sites+of+RNA+polymerase+II+stalling+by+interaction+with+Spt5.">Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.</a> Cell. 143, 122-133 (2010).</li><li>Lee-Theilen, M., Matthews, A. J., Kelly, D., Zheng, S., Chaudhuri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CtIP+promotes+microhomology-mediated+alternative+end+joining+during+class-switch+recombination.">CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.</a> Nat. Struct. Mol. Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <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+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

没有利益冲突的声明。

如前所述，多元化的抗体细胞株模型已经成为一种流行的出发点，以确定新的蛋白质，抗体多样化的影响在不同的步骤。我们在座的使用在拉莫斯B细胞病毒感染是击倒或过度表达的蛋白质的方法，然后检查SHM影响。 对于这些研究中，我们利用WT拉莫斯和援助您好拉莫斯细胞。野生型细胞可用于研究目标或表达的援助以及援助产生病变的修复，涉及的因素，而?...

推荐试剂-其中大部分来自其他厂商的同类产品，可代替 。 <table border="1" ><tbody><tr><th>试剂名称</th><th >公司</th><th>目录编号</th><th>评论</th></tr><tr><td > 6，以及明确的TC处理的板</td><td >康宁</td><td> 3516 </td><td ></td></tr><tr><td > 10毫升BD露儿- Loksyringes </td><td >屋宇署医疗</td><td > 309604 </td><td ></td></tr><tr><td> 24孔清晰TC治疗板</td><td >康宁</td><td > 3526 </td><td></td></tr><tr><td > TC - 96明确的平底聚苯乙烯处理微孔板</td><td >康宁</td><td > 3596 </td><td ></td></tr><tr><td > 100毫米的TC处理的培养皿</td><td >康宁</td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc针头式过滤器，0.45微米</td><td >颇尔生命科学</td><td > 4604 </td><td ></td></tr><tr><td >洋菜</td><td > Teknova </td><td > A7777 </tD&gt; <td ></td></tr><tr><td >琼脂糖</td><td > GeneMate </td><td> E - 3120 - 500 </td><td ></td></tr><tr><td >氨苄青霉素</td><td>西格玛</td><td > A0166 </td><td> 100 mg / ml的水</td></tr><tr><td >屋宇署FACSCanto II流式细胞仪</td><td> BD公司</td><td ></td><td >或类似的</td></tr><tr><td> BD猎鹰圆底聚苯乙烯管</td><td> BD公司</td><td > 352054 </td><td >为流式细胞仪</td></tr><tr><td > BOSC 23细胞</td><td > ATCC </td><td > CRL - 11270 </td><td ></td></tr><tr><td > 二氧化碳培养箱37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM（贝科的修改鹰的介质） </td><td >西格玛</td><td> D6429 </td><td ></td></tr><tr><td > FBS（胎牛血清） </td><td >双子座生物产品</td><td > 100-106 </td><td ></td></tr><tr><td > FITC标记的α-大鼠CD90/mouse CD90.1抗体</td><td > BioLegend </td><td > 202503 </td><td > FITC标记α- Thy1.1 </td></tr><tr><td > FuGENE 6转染试剂</td><td >罗氏公司</td><td > 11814443001 </td><td></td></tr><tr><td > HEPES缓冲溶液</td><td > Invitrogen公司</td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA高保真DNA聚合酶</td><td > KAPA生物系统公司</td><td > KK2101 </td><td ></td></tr><tr><td > LB肉汤（肉汤 - 卢里亚溶原性）粉</td><td > DIFCO </td><td > 240230 </td><td ></td></tr><tr><td >团真相与和解委员会的shRNA细菌甘油</td><td >西格玛</td><td ></td><td> shRNA的载体</td></tr><tr><td > NCS（新生小牛血清） </td><td >双子座生物产品</td><td > 100-504 </td><td ></td></tr><tr><td> PBS（磷酸盐缓冲溶液） </td><td > Invitrogen公司</td><td> 70011 </td><td >在水中稀释至1X </td></tr><tr><td >聚乙烯α-人IgM抗体</td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS（青霉素，链霉素，谷氨酰胺溶液） </td><td >双子座生物 - P产品展示</td><td > 400-110 </td><td ></td></tr><tr><td>凝聚胺（hexadimethrine溴） </td><td >西格玛</td><td > 107689 </td><td > 10 mg / ml的水</td></tr><tr><td > PureYield质粒Midiprep系统</td><td > Promega公司</td><td > A2495 </td><td></td></tr><tr><td>嘌呤霉素</td><td>西格玛</td><td > P8833 </td><td > 250微克/毫升在水中。 </td></tr><tr><td > QIAquick凝胶提取试剂盒</td><td >凯杰</td><td > 28706 </td><td ></td></tr><tr><td>拉莫斯（RA 1）细胞</td><td > ATCC </td><td > CRL - 1596 </td><td ></td></tr><tr><td > RPMI - 1640培养基</td><td >西格玛</td><td > R8758 </td><td ></td></tr><tr><td >标二</td><td > Invitrogen公司</td><td > 18064-022 </td><td></td></tr><tr><td > SYBR快速定量PCR试剂盒</td><td > KAPA生物系统公司</td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA聚合酶</td><td > Invitrogen公司</td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO的助教克隆试剂盒</td><td >体外根</td><td > K4520 - 01 </td><td ></td></tr><tr><td > TRIZOL </td><td > Invitrogen公司</td><td > 15596-026 </td><td ></td></tr><tr><td >向导SV基因组DNA纯化系统</td><td > Promega公司</td><td > A2361 </td><td ></td></tr><tr><td > X - Gal的[5 - 溴-4 - 氯- 3 - indoyl -β- D - galatopyranoside] </td><td > Growcells </td><td > C - 5687 </td><td > 40毫克/毫升，在DMSO </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

由V（D）J重组产生的低亲和力抗体的B细胞开始他们的生活。然而，检测的病原体后，变量（五）地区的免疫球蛋白（Ig）基因突变约100,000倍以上的基因组的其余通过体细胞超突变（SHM），高亲和力的抗体 1,2 。此外，类开关重组（CSR）产生一种免疫反应，是需要一个特定的病原体不同的效应功能的抗体。 CSR和SHM发起激活诱导的胞苷脱氨酶（AID），deaminates在DNA胞嘧啶残基产生uracils。这些uracils都容易出错的修复途径形式处理，最终导致突变和重组1-3。 我们目前所了解的SHM和CSR的分子细节来自相结合的研究在小鼠原代细胞，细胞系，细胞- F稀土元素的实验。小鼠模型仍显示许多修复因子（如雄，MSH2，MSH6，Exo1，和聚合酶η）4-10的关键作用的基因敲除的黄金标准。然而，并非所有的基因都为淘汰赛研究适合。例如，几双链断裂修复蛋白敲除embryonically致命或损害B细胞发展11-14。此外，有时在SHM或企业社会责任的一种蛋白的具体功能可能掩盖淘汰赛造成全球的缺陷。此外，由于在小鼠实验中，可以长篇大论，改变细胞系中的单个基因的表达已经成为一种越来越受欢迎的第一步，鉴定和表征候选基因15-18。 拉莫斯- Burkitt淋巴瘤细胞系组成进行SHM -曾流行一种细胞系模型来研究SHM 18-24。拉莫斯细胞的一个优势是，他们有一个内置的方便半定量测量的SHM。野生型细胞表达IgM和，因为他们拿起突变，一些淘汰IgM的表达的突变。因此，化验IgM的荧光激活细胞扫描仪（FACS）损失提供了SHM水平的快速读出。 SHM的定量测量，可通过直接的抗体基因测序。 由于拉莫斯细胞难以转染，我们生产的稳定增加或降低一个人的基因的表达与逆转录酶病毒或慢病毒的结构，包含一个过度表达纸盒或一个短发夹RNA（shRNA），分别感染细胞的衍生产品。在这里，我们描述了我们如何感染拉莫斯细胞，然后利用这些细胞来调查SHM特定基因的作用（图1）。

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

我们描述了如何执行逆转录病毒或慢病毒感染的过度表达或shRNA包含人类拉莫斯的B -细胞系构造和如何衡量在这些细胞中的体细胞超突变。

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

assessing-somatic-hypermutation-ramos-b-cells-after-overexpression-or

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.2K 次观看.  Duke University. 我们描述了如何执行逆转录病毒或慢病毒感染的过度表达或shRNA包含人类拉莫斯的B -细胞系构造和如何衡量在这些细胞中的体细胞超突变。

视频: Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

科研

癌症研究

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

遗传学

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

免疫与感染

<meta charset="utf8"></head><body>使用野生型封闭 PCR 优化低频突变检测

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

<meta charset="utf8"></head><body>作者聚焦：推进癌症组织中低频突变的检测

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

生物学

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

医学

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

从小鼠佩耶的补丁中对Germinal中心B细胞JH4因特龙的体细胞突变分析

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

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在纯化造血干/祖细胞识别DNA突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

从小鼠佩耶的补丁中对Germinal中心B细胞JH4因特龙的体细胞突变分析