We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).

1.基因被困胚胎干细胞库建设使用piggyBac转座子<ol><li>准备原代小鼠胚胎成纤维细胞（PMEF）作为馈线ES细胞培养<ol><li>在37℃水浴中解冻丝裂霉素C灭活PMEF（5.0×10 6）中的一个小瓶内。转移细胞到5ml ES细胞培养基（含有15％FBS的DMEM，1000单位/ ml白血病抑制因子，为100μM非必需氨基酸，2mM谷氨酰胺，55μM2-巯基乙醇，和25单位/ ml青霉素/链霉素），和离心机在100×g离心5分钟。 </li><li>吸出上清液并将细胞重新悬浮在30ml的ES细胞培养基，接着通过分配成两个T25烧瓶（各5毫升）和两个100毫米板（每次10毫升）。在37℃，5％CO 2的24小时。 </li></ol></li><li>文化与扩大C9胚胎干细胞<ol><li>在37℃水浴中解冻C9 ES细胞中的一个管形瓶（2.5×10 6个细胞在0.5毫升）和传送细胞到5ml ES细胞培养基中。离心在100×g离心5分钟。 </li><li>吸出上清，重悬ES细胞在5ml ES细胞培养基，接着通过电镀到T25供给器烧瓶中。在37℃，5％的CO 2。每天更换ES细胞培养基。 </li><li>培养2天后的，ES细胞应成为〜80％汇合，并已准备好进行传代。用5毫升的PBS洗涤细胞（不含Ca 2+和Mg 2+），然后添加2.5 ml的预热0.25％胰蛋白酶-EDTA。在37℃下进行10分钟。添加2.5毫升ES细胞培养基停止胰蛋白酶;移液器上下15次，打破了细胞团块。离心在100×g离心5分钟。 </li><li>吸在4ml ES细胞培养基的上清，重悬细胞。转让1 ml的每个含9 ml的ES细胞培养基的100mm板单元;准备两个100毫米板。这是一个1:8的通道。每日用10ml ES细胞培养基喂养细胞。 </li></ol></li><li>准备96孔运送板的库<ol><li>如1.1.1所述，解冻的丝裂霉素-C 3小瓶灭活PMEF（各含5×10 6个细胞）和重悬的细胞从每个小瓶用33.3 ml的ES细胞培养基。结合的细胞，得到100ml的细胞悬浮液。 </li><li>每孔到10块96孔板板加入100μl细胞。这些96孔饲养平板应该转染C9 ES细胞用 PB座子之前来制备至少一天。 </li></ol></li><li> C9 ES细胞与PB-UPA载体电穿孔<ol><li> Trypsinize和使用自动细胞计数器计数膨胀的C9 ES细胞。用10ml PBS洗C9 ES细胞的各100-mm印板。加入8 ml的预热0.25％胰蛋白酶-EDTA和孵育在37℃进行10分钟。 </li><li>通过加入2ml ES细胞培养基停止胰蛋白酶。细胞悬液转移到15毫升管和移液管上下15次掰开细胞团块。立即离心，在100×g离心5分钟，随后抽吸上清液。 </li><li>悬浮细胞小号在10ml ES细胞培养基和计数使用血球的ES细胞。制备细胞悬浮液6 15毫升管各含5×10 6 ES细胞。离心在100×g离心5分钟。吸出上清，悬浮每个细胞沉淀于0.8ml PBS中。 </li><li>制备6电试管（4毫米的间隙），所述0.8 ml的细胞转移。向每个试管中，添加1微克的PB-UPA和20μgMPB转座（mPBase）;移液器向上和向下轻轻拌匀。把反应杯在冰上。使用该指数设定为250伏的电穿孔，500μF，和无穷欧姆执行电。 注意:通常情况下，时间常数为约10至12毫秒为成功电穿孔，其产生约1,500至2000 G418每5×10 6个细胞抗性转化。 </li><li>检索，集中转染的细胞含有98毫升ES细胞介质的T75烧瓶中。的细胞混合通过温和移液，然后转移到细胞的贮存器（50毫升）中。我们荷兰国际集团一个12通道移液器，（共10个），每孔转移100μl的细胞对预先制备的96孔饲养平板。在37℃，5％的CO 2。 </li><li> 24小时后，每天喂细胞用新鲜ES细胞培养基含有G418（150微克/毫升），选择基因捕获克隆。 注:通常每口井将包含大约十G418抗性克隆。当这些菌落生长到足够的大小（通常为5-7天），所述96孔板（库）将被复制到2台进行存储和选择，分别。库中的每个板被指定为"子库&#39;，所以在这种情况下，有10个分库。 </li></ol></li></ol> 2.库复制<ol><li>复制法师板<ol><li> 24-48小时前的G418抗性集落达到50-60％汇合，解冻丝裂霉素C灭活PMEF的另外三个小瓶到10块96孔板进料器（参见1.3节）。明胶IZE 10 96孔板通过孵育0.1％明胶（100μl/孔）在37℃下至少15 - 30分钟; O / N培养是可以接受的。 </li><li>通过胰蛋白酶消化制备的96孔板单细胞悬浮液。从96孔板抽吸培养基，用PBS等体积洗一次（不含Ca 2+，Mg 2 +的）。加50微升预热0.25％胰蛋白酶-EDTA的，在5％CO 2在37℃下进行10分钟。停止用50μlES细胞培养基的胰蛋白酶。掰开细胞团块通过上下抽吸与多道移液器15倍。 </li><li>转移50微升胰蛋白酶细胞悬浮液给料机板（主）已经含有150微升ES细胞培养基，其余50微升到糊化板（复制品）已经含有150微升ES细胞培养基的相应行的相应的行。过程单元的每一行以一个主服务器和副本板。孵育板（现含200微升çulture体积）在37℃，5％的CO 2 O / N。 注意:现在有20个96孔板中。 </li><li>第二天喂细胞用含有150微克/毫升G418的100微升新鲜ES细胞培养基。每天更换介质，直到井80-90％汇合。 </li></ol></li><li>冻结法师板<ol><li>当主板块都在80％汇合，如（参见2.1.2节）trypsinize细胞。 </li><li>取出板，并停止用50μl的2x冷冻介质（60％ES细胞培养基，20％胎牛血清，20％DMSO）中的胰蛋白酶反应。移液器上下10次，多道移液器，以确保冷冻介质的适当混合。 </li><li>把96孔板的密封发泡胶盒，并将其放置在-80℃冷冻长达四个月的存储。四个月就会有足够的时间来完成压力的选择，并确定从主井板含有目的克隆。 注:贮存在-80＃176的温度超过4个月的，将导致细胞活力下降。一个可利用冷冻管在96孔格式，可以被存储在液氮用于存储长时间的汽相。 </li></ol></li><li>准备从副本板DNA板和分库池<ol><li> 24-48小时之前进一步复制的DNA板和分库池，制备10的100mm板丝裂霉素C灭活PMEF对于子文库池，和10胶凝96孔板将DNA复制板。参见1.3节的一般步骤。 </li><li> Trypsinize的细胞在96孔复制平板（参见2.1.2节）。在这10分钟胰蛋白酶消化步骤，从DNA板抽吸明胶和用含有150微升ES细胞培养基G418（150微克/毫升）代替。另外，准备含有5ml ES细胞培养基为&#39;分库&#39;汇集到100毫米的喂料机板的贮存器。 </li><li>胰酶消化之后，停止尝试PSIN用50μlES细胞培养基一行在一个时间使用12通道移液器。移液器上下10次掰开细胞团块。 </li><li>转移50微升细胞悬浮液的DNA板的相应的行和剩余的50微升转移到含有5ml ES细胞培养基容器。在完成一个完整的96孔板后，吸100mm的喂料机板的介质向其中汇集细胞转移。这些集中起来的突变细胞被指定为"子库"。 </li><li>重复其余9 96孔板中。每日用含有ES细胞培养基G418（150微克/毫升）喂96孔的DNA板和100毫米池&#39;子文库"板。 注:现在有10块96孔&#39;DNA&#39;板，10 100毫米的"子库"板块。 </li></ol></li><li>冻结DNA板。当细胞中的DNA板是80-90％汇合时，吸出介质，用100洗细胞两次微升PBS中并储存在-20℃下购买基因组DNA分离6。这些DNA板块将通过PCR筛选，以确定其在"主"板对应得很好则包含有用克隆。 </li></ol> 3，多西环素诱导的纯合突变体<ol><li>冻结分库池和传代细胞的强力霉素治疗<ol><li> 24小时前，100毫米"子库"具有殖民地大到足以产生70-80％汇合，准备一套十糊化100毫米板为多西环素治疗板。 </li><li>准备每个子库的单细胞悬液（参见1.4.1节和1.4.2的详细信息）。转印5×10 6细胞到糊化100毫米板用强力霉素（1微克/毫升）;孵育在37℃，5％ 的 CO 2。冻结剩余非多西环素处理的子文库池以每小瓶5×10 6个细胞。 </li></ol></li><li>代细胞在存在强力霉素淘汰博莱霉素<ol><li>通道的分库1:在强力霉素（1微克/毫升）长达两周的存在8每2-3天，在此期间DOX诱导敲除博来霉素的将促进杂合性缺失中的细胞人口，允许代纯合突变体。 </li></ol></li></ol> 4.压力选择和耐药克隆的隔离<ol><li>除多西环素处理的细胞应力选择<ol><li> 24小时传代强力霉素处理的细胞为应力选之前，制备10糊化150毫米板，发生在37℃，5％CO 2的湿润培养箱。 </li><li>准备每个分库通过胰蛋白酶消化的单细胞悬液（参见1.4.1节和1.4.2的详细信息）。种子6.6×10 6细胞到每个150毫米板在30毫升总培养体积，均匀分布的细胞。孵育细胞应激物（10μM百草枯或β巯基乙醇的遗漏，在含有7.5％热灭活的FBS）中7天，之后更换与正常ES细胞培养基，以允许存活细胞长成的菌落用于拾取ES细胞培养基。 </li><li>择:冻结左过度强力霉素处理的细胞以每小瓶5.0×10 6个细胞。 </li></ol></li><li>选择压力抗性菌落<ol><li>复原正常的媒体一个星期后，观察抗性菌落多少压力都存在。根据被拾起的菌落数，准备足够的井用丝裂霉素C灭活PMEF在96孔板1天相应提前（参见1.1节了解详细信息）。 </li><li>上的拾取，吸白天和替换介质中的96孔喂料机板用100μl新鲜ES细胞培养基，放置放回37℃，5％CO 2的湿润培养箱。此时，准备一个U型底96孔板用50μl0.25％胰蛋白酶-EDTA每孔。从150毫米压力选择板吸中，加测ePBS的QUAL量。 </li><li>微量设置为2微升，和&#39;挑&#39;存活菌落到U型底的胰蛋白酶含有孔，每孔采摘一个菌落。可以接受的是让在室温在板坐直至菌落的期望数量已经拾取。然后，孵育在U底板在37℃，5％CO 2的10分钟。 注:通常情况下，我们以1小时接96殖民地。 </li><li>停止胰蛋白酶反应一行与用多道移液器将50μlES细胞培养基一次。移液器上下15次，得到单细胞悬浮液和100μl的细胞转移到已含有100μl的ES细胞培养基的喂料机板的相应的行。完成转让整个板块。培养细胞O / N在200微升。用100μlES细胞培养基第二天早上，吸介质和更换。 </li></ol></li><li>复制的96孔板<ol><li>当拾取菌落的孔中是80-90％conflue核苷酸，复制成两个匹配板，其中之一用于DNA分离和另一个用于细胞备用。这些板块都胶凝，不包含丝裂霉素C灭活PMEF。 </li><li>吸出培养基，并用100微升PBS中。加入50微升的0.25％胰蛋白酶-EDTA到各孔，在37℃下进行10分钟。停止用150微升ES细胞培养基中胰蛋白酶，通过移液混合，并加入100μl细胞悬浮液转移到2 96孔板的相应的行。更改媒体每天。 </li><li>当板是80-90％汇合，冻结一组板作为"后备"（详情参见2.2节）。另一组板的如下所述的进一步扩大。 </li></ol></li><li>扩大细胞基因组DNA分离<ol><li> 96孔的DNA板（参照2.1.2细节）中制备单细胞悬浮液。 </li><li>从96孔板的每个单独孔用MICR转移细胞悬液至24孔板（共4个）的井opipette。确保有任何细胞无交叉污染，为"采摘"菌落"克隆"的起源。培养细胞在0.5ml ES细胞培养基，每天都在变化中，直到井80-90％汇合。 注:24孔板将留出足够的产量PCR分析基因组DNA。 </li></ol></li></ol> 5.确定的PB插入位被困基因<ol><li>从24孔板基因组DNA分离:裂解细胞，在24孔板和隔离无RNA基因组DNA。 </li><li>执行Splinkerette PCR（SpPCR），以产生序列片段作为所述7,8-侧翼该 PB座子整合位点。 注:SpPCR是五天的过程，但实际操作时间每天是最小的。 </li></ol>该突变克隆的6遗传分析<ol><li>找到主井和纯化目的克隆。 <ol><li>当PB整合位点已映射，设计了正向引物的整合位点的上游与PB反向引物（例如，PB3&#39;-1）8使用和筛选对应于该DNA的板相同的"子文库&#39;从该应力抗性克隆被挑选。期望找到唯一一个正以及整个96孔DNA板。从"大师"板解冻这一确切井24孔板上馈线和培养细胞，直到80-90％汇合。 注意:在这一点上，这孔包含的基因捕获克隆的混合物。 </li><li>当24孔板为80-90％汇合，trypsinize细胞和板1000 ES细胞在100mm的喂料机板（确保单细胞悬浮液）。通道剩余的细胞馈线扩张T25培养瓶中。从冻结的T25瓶中的细胞时，克隆是80-90％汇合。冻结每T25烧瓶作为后备4瓶。 </li><li>允许菌落生长7天，然后从中挑选96殖民地到96孔FE埃德尔板。参见4.2节采摘过程。复制版时准备好成为一个&#39;DNA&#39;板和冻结等为" 第二届-master"板（参见2.2节冻结）。 </li><li>允许菌落达到80-90％汇合。筛选该DNA板找到含有目的纯化的克隆孔中。扩大细胞从96孔2次-master板到24孔，并随后到T25培养瓶中（全都在馈线）。冻结每T25瓶中4瓶。 注意:在这一点上，该基因陷获细胞系是克隆，没有被暴露于应激物，并适合于鼠标的生产。 </li></ol></li><li>确定PB插入的数目:解冻压力耐药细胞株的一小瓶和扩大细胞的糊化T25烧瓶中。分离基因组DNA，并检查了PB转座子的拷贝数。这可以通过qPCR靶向新霉素基因来实现。 </li><li>测量基因表达水平:CONFI后中最差有在感兴趣的细胞系的单一的PB整合事件，测量，通过RT-qPCR的使用特定的Taqman探针为每个基因的捕获基因的表达水平。 </li><li>序列突变的mRNA:使用上游引物退火到外显子和下游引物退火的基因陷阱盒的剪接受体序列生成的cDNA的扩增子通过PCR。序列中的cDNA片段，以确认该预期拼接发生在捕获的基因。 </li><li>再动员piggyBac转座子来测试被困基因和抗应激能力之间存在因果关系。 <ol><li> 24-48小时前，解冻细胞在一个T25瓶中PB再活化，解冻和板DR4馈线和两个100毫米板。至少一天后DR4馈线镀到T25培养瓶中，解冻含有该 PB插入该细胞系中的一个小瓶和板的细胞在含DR4馈线的T25培养瓶中。生长中的细胞ES细胞培养基48小时，每天都在变化中。 </li><li>制备ES细胞的单细胞悬浮液。计数细胞用自动细胞计数仪，和5.0×10 6 ES细胞转移到15毫升管。离心机在100×g离心，并将细胞重新悬浮在800微升PBS中。该悬浮液转移至4mm杯。 </li><li>加入20微克mPBase 普罗小杯中，用移液混合，电穿孔细胞（参见1.4节了解详细信息）。分发转染的细胞到两个100毫米DR4饲养平板。培养细胞O / N在ES细胞培养基。 </li><li>第二天早上，喂细胞ES培养基用1微克/毫升嘌呤霉素和培养细胞48小时。撤回嘌呤选择，并继续培养细胞5天。当菌落采摘足够大，挑到一个96孔馈线板（参见4.2节）。 </li><li>当拾取的细胞已至80-90％汇合，分割板的1/6到的DNA板和1/6的复吃成G418板，留下2/3 RDS的细胞在原板被冻结（参见第2.2冻结）。 </li><li>培养该DNA板在购买DNA分离ES细胞培养基，和G418的板在150微克/毫升G418的测试对于新霉素抗性的丧失。威尔斯与72-96小时，在含G418培养后无活菌表明该基因捕获PB转座子成功的再活化。 </li><li>在&#39;脱氧核糖核酸"板中的相应孔将通过PCR进行筛选，以证实该 PB座子已被删除。当DNA板是90％汇合，隔离在96孔板格式 4的基因组DNA进行PCR筛选的野生型序列的恢复和不存在新的IRES的，以确认返原的装置。 </li><li>解冻从主板的匹配孔中，以24孔板并膨胀至T25烧瓶（所有与馈线）。冻结每T25瓶4瓶作为后备。 </li></ol></li></OL&gt; 7.代小鼠突变的<ol><li>恢复耐性ES细胞从主平板，并将其用于胚胎注射，以产生嵌合体。约15-20的ES细胞注射到个体中的C57BL / 6胚泡。传输每只小鼠囊胚15到假孕的女性（株ICR）子宫。交配雄性嵌合体（由ES细胞的特异性刺鼠毛色表示）从垃圾中回收的几个处女，C57BL / 6J雌性来测试种系传递，并建立突变小鼠的殖民地。 </li><li>隔离并测试尾部皮肤成纤维细胞，从F1的突变小鼠和野生型同窝出生的活性氧（ROS）的水平和耐百草枯9。 </li></ol>

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The authors have nothing to disclose.

Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants a...

长寿与抗逆性的亲密关系。在一般情况下，长寿命的物种通常显示增加的阻力向着多重压力，例如过氧化氢 ​​，百草枯（PQ），紫外线，热，和重金属1,2。与此相反，对压力增加的灵敏度趋向于预测寿命缩短和/或更多的疾病倾向的表型。抗氧化剂的清除途径早已推测在赋予抗逆性的动物中发挥重要作用。然而，除少数例外，从各种转基因动物的与各种抗氧化剂的酶（例如，超氧化物歧化酶）的操作的研究表明，增加的氧化剂清除酶的水平没有增加的寿命或健康跨度3。这些数据表明，在应力抗性性状在长寿命的动物始终观察由其他细胞途径尚未被揭露介导的。 我们采取了一个不带偏见向前遗传的方式来确定的基因，其在突变，可以赋予胁迫抗性表型中培养的胚胎干细胞（ES）细胞。 ES细胞提供在这项研究中两个主要的优点:（1）复杂的遗传操作可用于修改ES细胞的基因组中;和（2）的任何应力耐性ES细胞从屏幕中回收可直接用于鼠标的生产，从而允许快速翻译成整个动物研究来测量寿命和健康跨度。 在本报告中，我们描述了使用C9胚胎干细胞系，其中脂双层等位基因控制下由一个四环素响应元件。多西环素（DOX）治疗短暂关闭，博莱霉素导致姐妹染色单体交换的增加事件的表达。这种短期博来霉素敲除允许的杂人口内纯合突变的产生，使隐性突变为抗逆性可在筛选过程中被捕获。我们也描述了使用座子piggyBac（PB）作为转座子诱变随机插入 ​​聚-A捕获磁带（PB-UPA）变异基因在基因组中。细胞与基因由聚甲陷阱的破坏成为G418抗性和可被回收，这样的基因陷阱突变（基因陷阱库）的集合可以作出，并随后筛选突变体克隆是耐应力。 应力从选择回收的抗性克隆可以被相当迅速特征在于分子技术中插入的数目（定量PCR）的方面，插入部位（splinkerette PCR），被破坏的基因（BLAST）的身份，并且其表达水平（ RT-qPCR的）。 该 PB插入可以通过MPB转座的瞬时表达在克隆被重新活化用来恢复野生型DNA序列，从而测试应力阻力的损失。这些是有效的方法来确认突变的因果关系，应事先做昂贵的鼠标生产。以往的研究表明，细胞暴露在压力失去了他们的多能性4,5。因此，在该协议中，一个副本集突变的细胞，这将不能与压力处理的保存是成功小鼠生产的关键。 我们的实验室已经设计了C9胚胎干细胞系和PB-UPA载体，两者都可以应要求其他调查。这里所报告的协议将由从头文库基因束缚ES细胞用PB-UPA（图1A）中，然后影印和应力选以分离应力抗性克隆（ 图1B）的产生开始。我们证明百草枯，在细胞内一种强力游离自由基产生剂的选择。实际上，任何细胞毒性化合物或毒素，例如，ER 压力 （如，毒胡萝卜素和衣霉素），神经元氧化剂（例如，MPP +处理，6-羟基多巴胺，和鱼藤酮），热 ​​，和重金属 （如，镉，硒），可以适用于该方法以选择用于各个抗性突变体。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Vector&lt;/strong&gt;</td></tr><tr><td>PB-UPA</td><td></td><td></td><td></td></tr><tr><td>mPBase</td><td></td><td></td><td></td></tr><tr><td>mPBase&lt;sup&gt;Puro&lt;/sup&gt;</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td></tr><tr><td>500-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>250-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>50-ml Steriflip-GV filters</td><td>EMD Millipore</td><td>SE1M179M6</td><td></td></tr><tr><td>KO DMEM</td><td>Life Technologies</td><td>10829-018</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>FBS</td><td>Tissue Culture Biologicals</td><td>104</td><td></td></tr><tr><td>Heat Inactivated FBS</td><td>Sigma-Aldrich</td><td>F4135-500</td><td></td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140148</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Methyl Viologen dichloride (Paraquat)</td><td>Sigma-Aldrich</td><td>856177</td><td></td></tr><tr><td>Dimethyl Sulphoxide Hybri-MAX</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>EmbryoMAX 0.1% gelatin</td><td>EMD Millipore</td><td>ES006B</td><td></td></tr><tr><td>DPBS/Modified</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>T25 Flask</td><td>Corning</td><td>353108</td><td></td></tr><tr><td>T75 flask</td><td>Corning</td><td>353135</td><td></td></tr><tr><td>100-mm&amp;nbsp; plate</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>150-mm&amp;nbsp; plate</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>96-well&amp;nbsp; plate</td><td>Corning</td><td>3585</td><td></td></tr><tr><td>96-well U-bottom plate</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>50-ml reservoir&amp;nbsp;</td><td>Corning</td><td>4870</td><td></td></tr><tr><td>15-ml tubes</td><td>VWR International, LLC</td><td>82050-276</td><td></td></tr><tr><td>Primary Mouse Embryonic Fibroblasts</td><td>EMD Millipore</td><td>PMEF-NL</td><td></td></tr><tr><td>DR4 Mouse Embryonic Fibroblasts</td><td>Applied StemCell</td><td>ASF-1001</td><td></td></tr><tr><td>Mitomycin C</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP25312</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Life Technologies</td><td>11811-023</td><td></td></tr><tr><td>Doxycycline</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP26531</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td>377267</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5702</td><td></td></tr><tr><td>TC10 cell counter</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting Slides (for TC10)</td><td>Bio-Rad</td><td>1450011</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electroporation&lt;/strong&gt;</td></tr><tr><td>Gene Pulser Xcell</td><td>Bio-Rad</td><td>1652611</td><td></td></tr><tr><td>Gene Pulser Cuvettes (4&amp;#160;mm gap)</td><td>Bio-Rad</td><td>1652088</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Molecular Biology&lt;/strong&gt;</td></tr><tr><td>Thermal Cycler</td><td>Eppendorf</td><td>Mastercylcer ep Gradient&amp;nbsp; S</td><td></td></tr><tr><td>Puregene Core kit B</td><td>Qiagen</td><td>158745</td><td></td></tr><tr><td>Topo-TA Cloning kit</td><td>Life Technologies&amp;nbsp;</td><td>450030</td><td></td></tr><tr><td>High Capacity cDNA synthesis kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>NaCl</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP358-212</td><td></td></tr><tr><td>100% ethanol</td><td>Decon Laboratories, Inc.</td><td>2716</td><td></td></tr><tr><td>Double Processed Tissue Culture Water</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Sau3A1</td><td>New England BioLabs</td><td>R0169L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>EcoRV</td><td>New England BioLabs</td><td>R3195S</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;96-well Lysis Buffer (Ramires-Solis &lt;em&gt;et al.&lt;/em&gt; 1992)&lt;/strong&gt;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher BioReagents&amp;nbsp;</td><td>A144-212</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L5777</td><td></td></tr><tr><td>Proteinase-K</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1700</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electrophoresis&lt;/strong&gt;</td></tr><tr><td>Mini-Sub Cell GT</td><td>Bio-Rad</td><td>170-4469EDU</td><td></td></tr><tr><td>LE Agarose&amp;nbsp;</td><td>GeneMate</td><td>E3120500</td><td></td></tr><tr><td>Ethidium Bromide&amp;nbsp;</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1302</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>New England BioLabs</td><td>N3231S</td><td></td></tr><tr><td>1 Kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>2-log DNA Ladder</td><td>New England BioLabs</td><td>N3200L</td><td></td></tr></tbody></table>

forward genetic approach to uncover stress resistance genes in mice &#8212; a high-throughput screen in es cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

在典型的诱变实验中，转染由共3×10 7个ES细胞用PB-UPA和MPB转座的。在两个独立的实验中产生的基因捕获的ES细胞的数目总结在表1中。基因包封的效率是约0.04％。合并后的基因陷阱库包含22,400独立的突变体，小鼠基因组的约一个全覆盖（23,000编码基因）。过度的覆盖可以通过反复诱变产生更多的突变体和/或大规模的进程来实现。 因为基因陷阱库包含随机?...

Watch this Scientific Journal Video about Forward Genetic Approach to Uncover Stress Resistance Genes in Mice Ã¢â‚¬â€ A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

应力性的标志长寿之一，已知是基因支配。在这里，我们开发了一个不带偏见的高通量方法筛选，赋予抗逆性ES细胞以开发小鼠模型长寿研究突变。

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

forward-genetic-approach-to-uncover-stress-resistance-genes-mice-high

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  University of Colorado Denver. 应力性的标志长寿之一，已知是基因支配。在这里，我们开发了一个不带偏见的高通量方法筛选，赋予抗逆性ES细胞以开发小鼠模型长寿研究突变。 

视频: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

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 ...

科研

遗传学

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

发育生物学

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

生物学

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

用于鉴定裂殖酵母多拷贝抑制因子的遗传筛选

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

通过捕获Hi-C 破 译高分辨率3D染色质组织

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

摘要

探索更多视频

此视频中的章节

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

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

使用冷冻胚胎进行转基因小鼠高效生产

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

基于蛋白质的继承在酿酒酵母中的高通量筛选

C. Elegans生理应激反应的测量

用于鉴定裂殖酵母多拷贝抑制因子的遗传筛选

通过捕获Hi-C 破译高分辨率3D染色质组织

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

摘要

探索更多视频

此视频中的章节

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

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