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>

<ol>
	<li>Johnson, T. E., 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=Longevity+genes+in+the+nematode+Caenorhabditis+elegans+also+mediate+increased+resistance+to+stress+and+prevent+disease.">Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease.</a> J Inherit Metab Dis. 25, 197-206 (2002).</li><li>Murakami, S., Salmon, A., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+stress+resistance+in+cells+from+long-lived+dwarf+mice.">Multiplex stress resistance in cells from long-lived dwarf mice.</a> Faseb J. 17, 1565-1566 (2003).</li><li>Perez, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+overexpression+of+major+antioxidant+enzymes+does+not+extend+the+lifespan+of+mice.">The overexpression of major antioxidant enzymes does not extend the lifespan of mice.</a> Aging Cell. 8, 73-75 (2009).</li><li>Martin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+mice+to+test+the+oxidative+damage+theory+of+aging.">Genetic engineering of mice to test the oxidative damage theory of aging.</a> Ann NY Acad Sci. 1055, 26-34 (2005).</li><li>Chick, W. S., Drechsel, D. A., Hammond, W., Patel, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+mutant+phenotypes+from+ES+cells+to+adult+mice.">Transmission of mutant phenotypes from ES cells to adult mice.</a> Mamm Genome. 20, 734-740 (2009).</li><li>Ramirez-Solis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+microextraction:+a+method+to+screen+numerous+samples.">Genomic DNA microextraction: a method to screen numerous samples.</a> Anal Biochem. 201, 331-335 (1992).</li><li>Uren, A. G., 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=A+high-throughput+splinkerette-PCR+method+for+the+isolation+and+sequencing+of+retroviral+insertion+sites.">A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.</a> Nat Protoc. 4, 789-798 (2009).</li><li>Wang, W., Bradley, A., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piggyBac+transposon-based+genome-wide+library+of+insertionally+mutated+Blm-deficient+murine+ES+cells.">A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells.</a> Genome Res. 19, 667-673 (2009).</li><li>Chick, W. 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=Screening+for+stress-resistance+mutations+in+the+mouse.">Screening for stress-resistance mutations in the mouse.</a> Front Genet. 5, 310 (2014).</li><li>Salmon, A. B., 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=Fibroblast+cell+lines+from+young+adult+mice+of+long-lived+mutant+strains+are+resistant+to+multiple+forms+of+stress.">Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress.</a> Am J Physiol Endocrinol Metab. 289, 23-29 (2005).</li><li>Skarnes, W. C., 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=A+public+gene+trap+resource+for+mouse+functional+genomics.">A public gene trap resource for mouse functional genomics.</a> Nat Genet. 36, 543-544 (2015).</li><li>Baker, K. E., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense-mediated+mRNA+decay:+terminating+erroneous+gene+expression.">Nonsense-mediated mRNA decay: terminating erroneous gene expression.</a> Curr Opin Cell Biol. 16, 293-299 (2004).</li><li>Shigeoka, T., Kawaichi, M., Ishida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+nonsense-mediated+mRNA+decay+permits+unbiased+gene+trapping+in+mouse+embryonic+stem+cells.">Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.</a> Nucleic Acids Res. 33, e20 (2004).</li><li>Symula, D. J., Zhu, Y., Schimenti, J. C., Rubin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+annotation+of+mouse+mutations+in+embryonic+stem+cells+by+use+of+expression+profiling.">Functional annotation of mouse mutations in embryonic stem cells by use of expression profiling.</a> Mamm Genome. 15, 1-13 (2004).</li></ol>

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 border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vector</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PB-UPA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBasePuro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU05RE</td>
		</tr>
		<tr>
			<td>250-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU02RE</td>
		</tr>
		<tr>
			<td>50-ml Steriflip-GV filters</td>
			<td>EMD Millipore</td>
			<td>SE1M179M6</td>
		</tr>
		<tr>
			<td>KO DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Tissue Culture Biologicals</td>
			<td>104</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>EMD Millipore</td>
			<td>ESG1107</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140148</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
		</tr>
		<tr>
			<td>Methyl Viologen dichloride (Paraquat)</td>
			<td>Sigma-Aldrich</td>
			<td>856177</td>
		</tr>
		<tr>
			<td>Dimethyl Sulphoxide Hybri-MAX</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
		</tr>
		<tr>
			<td>EmbryoMAX 0.1% gelatin</td>
			<td>EMD Millipore</td>
			<td>ES006B</td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>Corning</td>
			<td>353108</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>353135</td>
		</tr>
		<tr>
			<td>100-mm&#160; plate</td>
			<td>Corning</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>150-mm&#160; plate</td>
			<td>Corning</td>
			<td>430599</td>
		</tr>
		<tr>
			<td>96-well&#160; plate</td>
			<td>Corning</td>
			<td>3585</td>
		</tr>
		<tr>
			<td>96-well U-bottom plate</td>
			<td>Corning</td>
			<td>3799</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3526</td>
		</tr>
		<tr>
			<td>50-ml reservoir&#160;</td>
			<td>Corning</td>
			<td>4870</td>
		</tr>
		<tr>
			<td>15-ml tubes</td>
			<td>VWR International, LLC</td>
			<td>82050-276</td>
		</tr>
		<tr>
			<td>Primary Mouse Embryonic Fibroblasts</td>
			<td>EMD Millipore</td>
			<td>PMEF-NL</td>
		</tr>
		<tr>
			<td>DR4 Mouse Embryonic Fibroblasts</td>
			<td>Applied StemCell</td>
			<td>ASF-1001</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP25312</td>
		</tr>
		<tr>
			<td>Geneticin (G418)</td>
			<td>Life Technologies</td>
			<td>11811-023</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP26531</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>377267</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5702</td>
		</tr>
		<tr>
			<td>TC10 cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Slides (for TC10)</td>
			<td>Bio-Rad</td>
			<td>1450011</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell</td>
			<td>Bio-Rad</td>
			<td>1652611</td>
		</tr>
		<tr>
			<td>Gene Pulser Cuvettes (4 mm gap)</td>
			<td>Bio-Rad</td>
			<td>1652088</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercylcer ep Gradient&#160; S</td>
		</tr>
		<tr>
			<td>Puregene Core kit B</td>
			<td>Qiagen</td>
			<td>158745</td>
		</tr>
		<tr>
			<td>Topo-TA Cloning kit</td>
			<td>Life Technologies&#160;</td>
			<td>450030</td>
		</tr>
		<tr>
			<td>High Capacity cDNA synthesis kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP358-212</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2716</td>
		</tr>
		<tr>
			<td>Double Processed Tissue Culture Water</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
		</tr>
		<tr>
			<td>Sau3A1</td>
			<td>New England BioLabs</td>
			<td>R0169L</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202T</td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England BioLabs</td>
			<td>R3195S</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Lysis Buffer (Ramires-Solis et al. 1992)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher BioReagents&#160;</td>
			<td>A144-212</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>L5777</td>
		</tr>
		<tr>
			<td>Proteinase-K</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1700</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT</td>
			<td>Bio-Rad</td>
			<td>170-4469EDU</td>
		</tr>
		<tr>
			<td>LE Agarose&#160;</td>
			<td>GeneMate</td>
			<td>E3120500</td>
		</tr>
		<tr>
			<td>Ethidium Bromide&#160;</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1302</td>
		</tr>
		<tr>
			<td>100 BP DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3231S</td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3232S</td>
		</tr>
		<tr>
			<td>2-log DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3200L</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 â€” A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#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

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

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 &#8212; A High-throughput Screen in ES Cells

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...