在本协议中所述的工作成为可能，通过与美国加州再生医学研究所（CIRM）的协作联盟的一部分，由维多利亚州政府资助。

所有过程都在无菌的层流罩中进行。所有介质和溶液平衡至37℃，除非另有规定。 1，扩展人类的产品分成合同（人类胚胎干细胞和iPS细胞，WA-09本议定书中所使用）的转注意:这是没有必要扩大对基质胶或Geltrex人类胚胎干细胞。人类胚胎干细胞展开对小鼠胚胎成纤维细胞（MEF中），可用于电或核转遵循MEF去除步骤（见第2.8节）。 <ol><li>制备1×100毫米培养皿和DR4的MEF中的2.0×10 4个细胞每cm 2在MEF媒体1×75平方厘米烧瓶中（ 见表1）上的菜肴预先涂有0.1％w / v的明胶。 注意:CF1，可以使用BLK6或MF1的MEF，但是，它建议抗生素有抗药性的MEF线被使用， 例如，DR410。 </li><li>第二天，通过胚胎干细胞到100毫米的菜预镀在1.1节准备的MEF。要做到这一点ASPI从培养的菜肴速率的hESC介质，洗的hESC与Ca2 + / Mg2 +的-free Hanks&#39;平衡盐溶液（HBSS）中，于37℃/ 5％CO 2在潮湿的培养箱中添加分散酶（ 见表2为适当体积）和地点。继3-5分钟孵化，吸分散酶，轻轻地洗1 - 2次的HBSS删除的MEF。添加人类胚胎干细胞介质（ 见表3），用细胞刮刀，或5毫升吸管轻轻刮落掉的培养皿的表面。 </li><li>收集悬浮在培养皿中的菌落片段入15ml离心管中。冲洗培养皿用人类胚胎干细胞的媒体和转移到15ml试管中。小心不要打破团块成单个细胞或小片段。 </li><li>离心5分钟，在160 XG以沉淀细胞集落的碎片。 </li><li>吸出上清液。挖掘15ml试管底部用手指拧松落碎片颗粒。根据分流比在人类胚胎干细胞介质轻轻悬浮细胞集落的碎片（normall4和1:8为宜）雅间1分流比。要格外小心，不要使团块成单个细胞或小片段，因为这会影响replating效率。 </li><li>此前replating人类胚胎干细胞碎片上的MEF用HBSS洗1X。慢慢地将适量含于人类胚胎干细胞介质的hESC片段的细胞悬浮液（ 见表2）添加到菜预镀的MEF。 </li><li>将培养皿架子上的孵化器，慢慢地前后移动和左右摇晃均匀分布的殖民地片段。 </li><li>每天更换人类胚胎干细胞介质。 </li><li>为了准备由MEF（MEF-CM）条件培养基洗75 平方厘米瓶预镀的MEF 1个用HBSS。人类胚胎干细胞中添加媒体。以下24个小时，收集条件培养基，换上新鲜的人类胚胎干细胞介质。每天最多12天重复。存储的MEF-CM在4℃下直到需要。 </li></ol> 2，准备人类胚胎干细胞的转染<ol><li>观察人类胚胎干细胞的生长每天在显微镜下。当菌落变成40-50％汇合时，板3×100毫米培养皿中，每平方厘米2.0×10 5的MEF。 </li><li>当菌落变成60 - 70％汇合，他们准备被转染。 &#39;新郎&#39;殖民地去除分化的殖民地，殖民地的核心。 注意:由于我们相信更高的转染和当细胞处于对数期生长靶向效率发生，故建议电穿孔进行之前的hESC达到≥70％汇合。 </li><li>用HBSS洗涤人类胚胎干细胞克隆，并添加的Accutase。 注意:这是没有必要预先治疗人类胚胎干细胞与ROCK抑制剂Y-27632时，在步骤2.7以Y-27632孵育会发生如足以抑制</li><li>孵育10 - 20分钟，在37℃/ 5％CO 2，直至菌落呈现单个细胞在显微镜下的观察。磨碎细胞以1毫升吸管。 </li><li>添加人类胚胎干细胞介质和过滤到15毫升锥形管使用40微米的细胞straineR以除去细胞团块。 </li><li>离心机在160×g离心5分钟。重悬细胞沉淀在新鲜胚胎干细胞介质，并重复以除去任何残留的Accutase溶液。 </li><li>重悬的细胞在胚胎干细胞介质含有10μM的Y-27632，总细胞悬浮液转移到100mm培养皿预先涂有明胶。 </li><li>在37°C / 5％CO 2的至少30分钟。 </li><li>在此期间的MEF将坚持以明胶涂层菜。收集使用1毫升吸管非粘附的hESC成新鲜15ml离心管中，并确定使用血球细胞密度。 </li></ol> 3，电穿孔胚胎干细胞<ol><li>使用0.22微米过滤器，并加入10纳克/ ml重组人成纤维细胞生长因子2（rhFGF2）过滤器CM-MEF媒体。 </li><li>传输距离2.9节7.5×106个胚胎干细胞到一个新的15毫升锥形管和离心160×g离心5分钟。 </li><li>在此期间，成立了电穿孔:选择程序的指数ELEctroporation并设置以下参数，电压:250伏，容量:500μF，和阻力:∞-。 </li><li>吸出上清液，并于0.8ml冰冷却的0.22微米过滤的HBSS中重悬细胞沉淀。将溶液转移到无菌的0.4cm电比色皿。 </li><li>添加电视hPITX3进靶向构建（30微克DNA），并用200微升移液器轻轻混匀。在冰上孵育5分钟。在这段时间删除PITX3 ZFN表达的一个分装解冻冰。当电比色皿解冻转移7.5微升ZFN基因与DNA /细胞混合物。用200微升移液器轻轻混匀。 注意:作为目标的轨迹是有助于靶向效率的主要因素。基因座编码沉默基因，不表达于胚胎干细胞的基因，如PITX3是难以用常规的同源重组6进行定位。因此，该协议利用定制的ZFN为人类PITX3基因诱导的DNA双链break。 </li><li>通过电穿孔以下的电穿孔仪屏幕上的说明进行操作。 </li><li>用200微升吸管从电比色皿的内容传送到一个​​15毫升的试管含有10ml人类胚胎干细胞介质。与每个时间，并转移到15ml试管中加入200μl人类胚胎干细胞介质的两次冲洗比色皿。会出现"粘液状"碎片组成的DNA和蛋白从裂解的细胞将细胞存活产生不利影响的。离心机在160×g离心5分钟以除去这些碎屑。 </li><li>吸出上清液，并轻轻拍打细胞沉淀松动。重悬在30毫升补充的MEF-CM含有10微米Y-27632和replate上预镀用的MEF 3×100毫米的菜肴。 </li><li>每天更换介质。在第2天，Y-27632可抽出并生长的细胞中只有人类胚胎干细胞的介质。 </li><li>后2-3天的人类胚胎干细胞应该是50-70％汇合。在这一点上开始抗生素选择（ 例如，0.5微克/毫升嘌呤霉素）。 </li><li>保持SEL挠度通过每天增加新鲜胚胎干细胞/嘌呤霉素媒体。后〜7天小菌落应该是可见的。如果不使用抗生素有抗药性的MEF，MEF中通过每平方厘米增加一个额外的1.0×10 4个细胞的补充。 </li></ol> 4，采摘转基因人类胚胎干细胞克隆<ol><li>大约14 - 22天内选择（当殖民地已经达到约1毫米直径）准备板的扩展和筛选。通过解冻基底膜或Geltrex的等分在4℃下至少5小时的开始。稀释按制造商的指示，并添加75微升至3.0×96孔板各孔中。通过在2.0×10 4个细胞每cm 2电镀的MEF制备3×48孔板中。孵育两个板在o / n 37℃/ 5％的CO 2。 </li><li>用在2毫升吸管的端部的21G针头切网格次日解剖菌落成16-25件。 </li><li>吸出基质胶从96孔板中，并替换为supplemented CM-MEF媒体。洗的MEF在48孔板1个用HBSS，并添加人类胚胎干细胞的介质。 </li><li>用200微升吸管轻轻转移解剖殖民地的三分之一到48孔板进行扩展。传送解剖菌落的其他三分之二到含有补充CM-MEF媒体对应的96孔板中。 </li><li>通过每日更换介质保持两板（补充CM-MEF培养基的96孔基质胶包被的平板和人类胚胎干细胞培养基包含的MEF 48孔板;都补充有0.5微克/毫升嘌呤霉素）。 </li><li>扩大直到96孔板100％汇合，然后开始通过PCR筛选。 </li></ol> 5，筛选和扩展阳性克隆<ol><li>要准备的基因组DNA（基于11法）由反相删除板所有媒体，然后涂抹到纸巾。 </li><li>冲洗各孔两次，用RT PBS（100μl/孔，每次洗涤），转化和印迹，作为步骤5.1。地方板在-80°C 2 F或1 - 2小时，细胞裂解援助（板可以无限期地保存这样的）。 </li><li>而细胞在-80℃下使烷基肌氨酸钠裂解缓冲液（ 见表4）（50μl/孔）。裂解细胞，暖板5分钟，在RT再吸移管的裂解缓冲液到每个孔中。封含有湿润纸巾湿度创建一个可密封的容器中的每个盘磁带，地方的边缘，并培育在o / n 60℃。 </li><li>孵育后加入150μl5M氯化钠10毫升-20℃的无水乙醇，混合均匀，并加入100微升，每孔（注:酒精会去阴天时NaCl的加入）。轻轻拍打板混合（不要吸管），并留下了至少30分钟，在室温。在此期间，溶液会清楚的DNA会沉淀。 </li><li>除去溶液如在步骤5.1，然后加入150微升70％乙醇中的每个孔中。轻轻翻转板和以前一样吸干。重复此步骤，进一步的2倍。 注:由于沉淀的DNA自然阿德水库于组织培养塑料它不会成为在这些洗涤步骤被撞出。） </li><li>最后一次洗涤后，短暂离心平板以最高速度来浓缩DNA到各孔然后空气干燥的在室温下（即37℃）下至少30分钟。 </li><li>添加40 - 50微升T0.1E液（pH8.0）（ 见表5）或TE（pH为8.0），向每个孔中并置于平板在65℃下进行1小时或在4℃CO / N使用用于PCR之前。通过轻轻敲击板的侧面重悬DNA。使用1-2微升的再悬浮的DNA / 10微升的PCR反应。 </li><li>使用Expand长模板PCR系统与hPITX3Ł臂根。 F和hPITX3Ļ臂GFPŕ引物进行克隆的初步筛选，在58℃退火，45秒延伸，35个循环。 </li><li>与Hyperladder 1KB在1％琼脂糖凝胶Electrophorese样本含有Gel​​Red在100伏45分钟，并通过紫外线照射检测阳性克隆。 </li><li>验证PCR阳性克隆，通过消化，从EXPA基因组DNAnded克隆（见下）用HindIII和杂交探针的5&#39;和3&#39;的载体同源臂，以Southern印迹（如先前所述 ）。通过PCR使用引物对hPITX3 5&#39;探针F和R，并hPITX3 3&#39;探针F和R，用EcoRI消化，并连接到一个通用的克隆载体中产生的探针。 </li><li>根据阳性克隆的数目，通过预镀用的MEF（2.0×10 4个细胞每cm 2）的制备2孔6孔板。 </li><li>第二天预处理阳性克隆在48孔板为10μm的Y-27632最少1小时的。 </li><li>用HBSS洗阳性克隆，并加入300μl的Accutase的。 </li><li>当菌落呈现单细胞，加入1 ml人类胚胎干细胞介质和收集到15毫升管。 </li><li>每天更换介质。在第2天，Y-27632可抽出并生长的细胞在人类胚胎干细胞的介质。 </li><li>当殖民地的70 - 80％融合，传代培养和冷冻的结肠其余纽约片段。 </li></ol>

<ol>
	<li>Nefzger, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmx1a+Allows+Context-Specific+Isolation+of+Progenitors+of+GABAergic+or+Dopaminergic+Neurons+During+Neural+Differentiation+of+Embryonic+Stem+Cells.">Lmx1a Allows Context-Specific Isolation of Progenitors of GABAergic or Dopaminergic Neurons During Neural Differentiation of Embryonic Stem Cells.</a> Stem Cells. 30 (7), 1349-1361 (2012).</li><li>Zeng, W. R., Fabb, S. R. A., Haynes, J. M., Pouton, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+periods+of+neural+induction+and+propagation+of+embryonic+stem+cell-derived+neural+progenitors+with+EGF+and+FGF2+enhances+Lmx1a+expression+and+neurogenic+potential.">Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential.</a> Neurochemistry International. 59 (3), 394-403 (2011).</li><li>Collin, J., Lako, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+putting+a+finger+on+stem+cell+biology:+zinc+finger+nuclease-driven+targeted+genetic+editing+in+human+embryonic+stem+cells.">Concise review: putting a finger on stem cell biology: zinc finger nuclease-driven targeted genetic editing in human embryonic stem cells.</a> Stem Cells. 29 (7), 1021-1033 (2011).</li><li>Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects:+zinc-finger+nucleases+as+gene+therapy+agents.">Progress and prospects: zinc-finger nucleases as gene therapy agents.</a> Gene Therapy. 15 (22), 1463-1468 (2008).</li><li>Urnov, F. D., 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=Highly+efficient+endogenous+human+gene+correction+using+designed+zinc-finger+nucleases.">Highly efficient endogenous human gene correction using designed zinc-finger nucleases.</a> Nature. 435 (7042), 646-651 (2005).</li><li>Hockemeyer, D., 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=Efficient+targeting+of+expressed+and+silent+genes+in+human+ESCs+and+iESCs+using+zinc-finger+nucleases.">Efficient targeting of expressed and silent genes in human ESCs and iESCs using zinc-finger nucleases.</a> Nature Biotechnology. 27 (9), 851-857 (2009).</li><li>Lombardo, A., 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=Gene+editing+in+human+stem+cells+using+zinc+finger+nucleases+and+integrase-defective+lentiviral+vector+delivery.">Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.</a> Nature Biotechnology. 25 (11), (2007).</li><li>Boch, J., Bonas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthomonas+AvrBs3+family-type+III+effectors:+discovery+and+function.">Xanthomonas AvrBs3 family-type III effectors: discovery and function.</a> Annual Review of Phytopathology. 48, 419-436 (2010).</li><li>Hockemeyer, D., 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=Genetic+engineering+of+human+embryonic+cells+using+TALE+nucleases.">Genetic engineering of human embryonic cells using TALE nucleases.</a> Nature Biotechnology. 29 (8), 731-734 (2011).</li><li>Tucker, K. L., Wang, Y., Dausman, J., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+mouse+strain+expressing+four+drug-selectable+marker+genes.">A transgenic mouse strain expressing four drug-selectable marker genes.</a> Nucleic Acids Research. 25 (18), 3745-3746 (1997).</li><li>Ram&#237;rez-Solis, R., Rivera-P&#233;rez, J., Wallace, J. D., Wims, M., Zheng, H., Bradley, A. <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> Analytical Biochemistry. 201 (2), 331-335 (1992).</li><li>Kawasaki, H., 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=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28 (1), 31-40 (2000).</li><li>Denham, M., Thompson, L. H., Leung, J., P&#233;bay, A., Bj&#246;rklund, A., Dottori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gli1+is+an+inducing+factor+in+generating+floor+plate+progenitor+cells+from+human+embryonic+stem+cells.">Gli1 is an inducing factor in generating floor plate progenitor cells from human embryonic stem cells.</a> Stem Cells. 28 (10), 1805-1815 (2010).</li><li>Smidt, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homeodomain+gene+Ptx3+has+highly+restricted+brain+expression+in+mesencephalic+dopaminergic+neurons.">A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons.</a> Proceedings of the National Academy of Sciences U.S.A. 94, 13305-13310 (1997).</li></ol>

作者什么都没有透露。

对记者的细胞系的产生提供了强有力的手段来跟踪，可视化和从人类胚胎干细胞来源的异质群体隔离感兴趣的细胞。然而，基因通过常规的同源重组靶向已被证明是非常低效的人类胚胎干细胞3。在这个协议中，我们描述了一个相对简单的技术采用广泛使用的目标向量的ZFN一起引入到RP的外显子的PITX3基因之一，在人类胚胎干细胞。我们手中的基因靶向系统是高效的，用含有正确的目...

当前的胚胎干细胞（ESC）分化的协议导致异质细胞群，特别是相对于特定的神经元表型的推导。因此，虽然文化包含所需的细胞表型，其他的神经元，非神经和未分化的细胞类型是经常存在1。这些特点限制了ESC键的应用程序源细胞来源的细胞替代疗法的使用和体外疾病模型。 遗传报告基因的细胞系提供了可视化，跟踪和隔离所关注的细胞的装置，只要该报告蛋白（RP）中的表达再现的内源性表达。使用的启动子紧接基因编码区上游的可用于推导粗遗传记者但这样的构建缺乏控制内源基因表达的精确调控元件。与此相反，同源重组提供了机会，确保安置计划的高保真表达。在过去，针对设计用于同源重组在感兴趣的特定基因的载体已被用于使用电穿孔作为DNA递送1,2的手段对目标在小鼠胚胎干细胞（mESCs分化）的RP。但是通过传统的同源重组报告细胞系的生成是非常低效的人类胚胎干细胞（胚胎干细胞），因此只被记录在极少数情况下（在3审查）。通过使用含有融合的霍 1核酸酶与特定地点的锌指结构，统称为锌指核酸酶（ZFN）的工程嵌合蛋白，DNA双链断裂可以在预先确定的基因组位点引入。当用同源性的DNA载体的DNA双链断裂的两侧添加，基因组位点可通过同源重组得到修复，从而使该DNA供体序列的掺入。该技术已被证明非常有用˚F或在两个人原代细胞4,5和6,7人类胚胎干细胞的基因组编辑。更近期的工作已经利用转录激活因子样效应物核酸酶（TALENS），所使用的植物转录因子的病原体如图8所示 ，位点特异性核酸酶9来帮助设计。 在下面的协议我们展示了人类胚胎干细胞报道细胞系的产生，含有同源靶向载体6用的ZFN对人垂体同源3（PITX3）轨迹在一起的EGFP的电穿孔。下列抗生素选择为2-3周，胚胎干细胞具有正确整合的DNA可以手动拾取，膨胀并通过PCR筛选最初，并随后通过Southern杂交验证。

<table><tbody><tr><td>Mouse Embryonic Fibroblasts (DR4)</td><td>GlobalStem</td><td>GSC-6004G</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G1393-100ML</td><td></td></tr><tr><td>HBSS, no Calcium, no Magnesium, no Phenol Red (HBSS)</td><td>Life Technologies</td><td>14175-095</td><td></td></tr><tr><td>Dispase</td><td>StemCell Technologies</td><td>7923</td><td></td></tr><tr><td>Cell Scraper</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A11105-01</td><td></td></tr><tr><td>Y27632</td><td>Axon Medchem</td><td>Axon 1683</td><td></td></tr><tr><td>Cell Strainer - 40 &amp;mu;m</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>33 mm - 0.22 &amp;mu;m filter</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>rhFGF2</td><td>R&amp;amp;D Systems</td><td>233-FB-025/CF</td><td></td></tr><tr><td>Electroporator</td><td>BioRad</td><td>165-2661</td><td></td></tr><tr><td>0.4 cm electroporation cuvette</td><td>BioRad</td><td>165-2088</td><td></td></tr><tr><td>Human TV-hPITX3-forward targeting construct</td><td>Addgene</td><td>31942</td><td></td></tr><tr><td>ZFN Kit; Human PITX3</td><td>Sigma-Aldrich</td><td>CKOZFN1050-1KT</td><td></td></tr><tr><td>Puromycin</td><td>InvivoGen</td><td>ant-pr-1</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>Geltrex</td><td>Life Technologies</td><td>A1569601</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>10566-016</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, Heat Inactivated, US Origin (FBS)</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td><td>Life Technologies</td><td>11320-033</td><td></td></tr><tr><td>KnockOut Serum Replacement (KSR)</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids&amp;nbsp; (NEAA)</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>&amp;szlig;-mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>N-Lauroylsarcosine sodium salt solution (Sarcosyl) (30%)</td><td>Sigma-Aldrich</td><td>61747</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/e9884?lang=en&amp;amp;region=US&quot;&gt;E9884&lt;/a&gt;</td><td></td></tr><tr><td>Trizma Hydrochloride</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Proteinase K solution (20 mg/ml)</td><td>Bioline Australia</td><td>BIO-37084</td><td></td></tr><tr><td>Expand Long Template PCR System</td><td>Roche Australia</td><td>11681834001</td><td></td></tr><tr><td>hPITX3 L arm gen. F (primer): 5&amp;rsquo; TGTCCTAAGGAGAATGGGTAACAGACA 3&amp;rsquo;</td><td>GeneWorks, Australia</td><td></td><td></td></tr><tr><td>hPITX3 L arm GFP R (primer): 5&amp;rsquo; ACACGCTGAACTTGTGGCCGTTTA 3&amp;rsquo;</td><td>GeneWorks, Australia</td><td></td><td></td></tr><tr><td>HyperLadder 1 kb&amp;nbsp;</td><td>Bioline Australia</td><td>BIO-33025</td><td></td></tr><tr><td>GelRed</td><td>Jomar Bioscience, Australia</td><td>41003</td><td></td></tr></tbody></table>

zinc-finger nuclease enhanced gene targeting in human embryonic stem cells

与当前的人类胚胎干细胞（ESC）的分化方案的一个主要限制是异质的细胞群的产生。这些培养物含有感兴趣的细胞，而且还污染了未分化的胚胎干细胞，非神经衍生物和其他神经元亚型。这限制了它们的使用在体外和体内应用中，如在体外模型用于药物开发的或细胞替代疗法。为了帮助解决这个问题，记者的细胞系，它提供可视化，跟踪和隔离感兴趣的细胞的方法，可以设计。然而，要实现这个在人胚胎干细胞通过常规的同源重组是极其低效的。这个协议描述了一种具有靶向垂体同源使用定制设计的锌指核酸酶，其引入位点特异性双链DNA断裂3（PITX3）位点在人胚胎干细胞，一起PITX3 - 绿色荧光蛋白特异性的DNA供体载体。以下的PITX3报道细胞系的产生，其然后可以使用发表的方案为在研究中使用的分化，例如在体外帕金森氏病模型或细胞替代疗法。

以下共电穿孔的定制设计PITX3锌指一对连同PITX3 - EGFP特异性DNA供体载体中，并随后嘌呤选择，阳性胚胎干细胞克隆，通过基因组PCR筛选（ 图1），首先检测到的。这些PCR阳性克隆与5&#39;和3&#39;特异性探针的Southern印迹杂交确认正确的定位外显子1的PITX3轨迹（ 图2），具有19％的效率。在图3所示的免疫荧光图像表明EGFP表达由PITX3子使用?...

Watch this Scientific Journal Video about Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells at JoVE.com

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

记者的细胞系提供了可视化，跟踪和隔离，从不同的人群感兴趣的细胞的方法。然而，基因靶向用常规的同源重组在人胚胎干细胞是非常低效的。在这里，我们将介绍使用锌指核酸酶增强同源重组靶向中枢神经系统中脑特异性转录因子PITX3基因与绿色荧光蛋白。

锌指核酸酶增强基因打靶的人胚胎干细胞

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

zinc-finger-nuclease-enhanced-gene-targeting-human-embryonic-stem

Research

JoVE Journal

Biology

10.8K 次观看.  Monash University. 记者的细胞系提供了可视化，跟踪和隔离，从不同的人群感兴趣的细胞的方法。然而，基因靶向用常规的同源重组在人胚胎干细胞是非常低效的。在这里，我们将介绍使用锌指核酸酶增强同源重组靶向中枢神经系统中脑特异性转录因子PITX3基因与绿色荧光蛋白。 

视频: Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

科研

发育生物学

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

使用无缝基因组编辑将点突变引入人类多能干细胞

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

遗传学

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

生物学

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

医学

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

小鼠基因组工程使用Designer核酸酶

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

<meta charset="utf8"></head><body>CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical ...

锌指核酸酶增强基因打靶的人胚胎干细胞

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转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

建立基因组编辑的人多能干细胞系:从定位到隔离

使用无缝基因组编辑将点突变引入人类多能干细胞

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

人类诱导多能干细胞的转染和Nucleofecting

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成

小鼠基因组工程使用Designer核酸酶

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失