Name: transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor
Uploaded: 2015-02-01T14:00:00.000+00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1.准备基底膜基质和塑料制品的涂装<ol><li> -20℃放置冷冻基底膜基质的库存在冰上解冻过夜，在4℃。 </li><li>解冻后，基底膜基质的吸移管2毫克等分入预冷的微量离心管中。直到需要℃，-20存储这些。 </li><li>为了准备基底膜基质涂层钢板，解冻一个等份冰上直到最后一块冰，在离心管消失（通常在〜2小时）。 </li><li>解冻后，基底膜基质添加至12毫升冷（4℃）的DMEM / F12，使基底膜基质涂布液。 </li><li>加入基底膜基质上解决了相应的培养容器。为一个6孔板，分配每孔1ml。摇动板以确保基底膜基质溶液完全覆盖每个孔中。 </li><li>封口膜密封基底膜基质涂层板/皿，孵育袋鼠米的温度在使用前1小时。备选地，2周涂层的内店铺基底膜基质包被的平板/菜在4℃下并使用。 注:添加额外的DMEM / F12向基底膜基质涂覆后的板/培养皿防止干燥。用4℃储存基底膜基质涂覆后的板/培养皿之前，将其放置在生物安全柜，并允许它达到室温至少30分钟。 </li><li>完全加入培养基和细胞的前抽吸基底膜基质。 </li></ol> 2.准备E8介质<ol><li>通过解冻E8补充过夜，在4℃下制备E8培养基。 </li><li>从500毫升的库存除去10毫升E8基础培养基并丢弃。 </li><li>吸取了整个10毫升小瓶E8补充直接进入490毫升E8基础培养基。不热情完全E8媒体在37℃水浴，因为温度反复波动会降低的bFGF在完井TE E8中。 </li><li> 2周另外补充的范围内使用完整的E8媒体。 </li></ol> 3.解冻iPS细​​胞的<ol><li>取下液氮和地点干冰冷冻iPS细​​胞的小瓶。 </li><li>迅速解冻在37℃的水浴的小瓶;漩涡在水浴小瓶，直到冰遗体只有一小片段。 </li><li>喷用70％乙醇和转移小瓶至生物安全柜中。 </li><li>加入1毫升室温E8介质滴加直接向小瓶中。 </li><li>使用2毫升吸管，转移细胞悬液滴加到9ml E8培养基在15ml锥形管中。摇动管经常以确保细胞和培养基迅速拌匀。 </li><li>离心细胞，在200×g离心5分钟。 </li><li>吸出上清液，和重悬细胞沉淀在E8的适当体积补充有10μM的Y-27632。 </li><li>添加细胞地下室适当数量MEMBRANê基质包被的孔，并将其放置在37℃，5％CO 2的培养箱中过夜。建议板至少0.2×10 6的iPSC每一个6孔板的井，使解冻后快速恢复。 </li></ol> 4.维护和iPS细胞的常规传代<ol><li>每天刷新E8媒体。 </li><li>监视形态和细胞的汇合与倒置显微镜。高品质的iPS细胞在生长具有明显的边界平整的殖民地;单个菌落拥有"铺路石样"外观。 </li><li>通道iPSCs的细胞，当他们到达〜70％汇合。 </li><li>通过加入0.9克NaCl和500μl的0.5M的EDTA至500ml的DPBS制备的EDTA传代方案。拌匀溶解NaCl和真空过滤消毒。暖在之前传代37℃水浴中传代溶液的等分试样。 </li><li>向通道，抽吸掉废培养基并用温水PAS等体积一次洗涤细胞saging解决方案。吸出物，以及移液管足够的EDTA传代溶液涂覆的细胞（每一个6孔板的孔1毫升）中。 </li><li>放置细胞的倒置显微镜下，观察的iPSC集落。孔殖民地和提高境内的外观应在2至5分钟变得明显。 </li><li>小心吸了EDTA传代的解决方案。 </li><li>用10毫升吸管，分配4毫升E8介质（如果使用6孔板）在高压下直接进入每个孔进行传代。 </li><li>收集的iPSC团块，并分裂成根据从1分流比阱的适当数量:8至1:12不要过度吸管，因为细胞团块解体将导致生存能力差。 </li><li>将板在培养箱中，并摇动板背面的往复和侧到另一侧的几次，以分散细胞。 </li></ol> 5.准备的MEF和iPS细胞的Tansfection的<ol><li> 48小时前transfectioN，通道的iPSC在〜1:6成四个或更多个井基底膜基质包被的6孔板的，使得它们将是70％汇合2天因此。 </li><li>第二天，解冻DR4的MEF成由DMEM（高葡萄糖），补充有10％FBS和1x MEM-NEAA的MEF培养基。 </li><li>板DR4的MEF成两个10厘米菜肴〜2×10 4个细胞/ cm 2孵育过夜，在37℃。 </li><li>在转染的当天，在iPS细胞进行转染前30分钟改变MEF培养基至E8补充有10μM的Y-27632。 </li><li>可选:如果iPSCs的转染后的流式细胞术分析是需要的，除去从4℃的基底膜基质涂覆后的板和放置在室温下。 </li><li>可选:4小时在转染之前，补充预染的iPSC培养用Y-27632以10μM的最终浓度。 </li></ol> 6.温和，细胞分离试剂治疗iPS细胞的转染次使用电穿孔系统<ol><li>从4℃除去P3的初级细胞的转染溶液，并允许它来室温〜30分钟。整个加入100μl补充添加到使用前的转染溶液。 </li><li>温暖柔和的细胞分离试剂在37℃水浴。 </li><li>得到AAVS1 TALENS（PZT-AAVS1-L1和PZT-AAVS1-R 1）和AAVS1-CAG-EGFP -20℃供体。 </li><li>从培养箱中取出的iPSC和用DPBS洗涤一次。 </li><li>加入1毫升平缓细胞解离试剂以每孔，并孵育iPSCs的在37℃下5分钟，或直至细胞大于50％的从培养容器中解离。 </li><li>吸取细胞上下用P1000吸管解离从培养容器中的任何剩余的细胞，并打散的iPSC团块几次。 </li><li>加2ml E8培养基到每个孔中，并且吸管上下数次用10毫升吸管给菲尔特呃分解细胞团块成单个细胞 注:转染效率显著下降，如果细胞团块不够分类。 </li><li>收集的iPSC在15ml锥形管中，并离心，在100×g离心3分钟。 </li><li>吸去上清液，并在E8介质的最小量重悬细胞。 </li><li>算使用的应用的一个重要污点诸如0.4％台盼蓝后血球细胞。确保细胞被充分分解，同时计数（1-3单元格每个"丛"）。 </li><li>分配3×10 6细胞到每个2的15毫升锥形管中，并再次降速，在100×g离心3分钟。 注:低速离心降低了细胞应激，并允许电前容易再悬浮iPS细胞的。 </li><li>电穿孔系统设置为对人胚胎干细胞系H9（程序的CB-150）的细胞类型特异性的程序。 </li><li>离心后，从CEL吸出上清液升颗粒。一个小球，加10微克的HR捐助作为对照样本。的其他粒料加10微克的人力资源供体，连同5微克每TALEN（PZT-AAVS1-L1和PZT-AAVS1-R 1）作为实验样品。 </li><li>重悬在100μl的P3的初级细胞的转染溶液各细胞沉淀，并转移到一个试管中。 </li><li>完成转染，并立即加入500μl室温E8培养基到每个试管中。 </li><li>转移的iPS细胞转染滴含步骤5.4准备DR4 MEF中一个10厘米的菜。 </li><li>可选:从每个实验添加数滴成基底膜基质包被的6孔板的一个孔中，如果流式细胞转染效率anaylsis是期望的。 </li><li>重复步骤6.15-6.17完成两个样本。次日，洗涤细胞用DPBS两次并切换培养基NutriStem，这似乎支持的iPSC培养上馈线优于E8。 </li><li> TRansient EGFP表达的峰在48到72小时后转染;根据需要评估转染效率。 </li></ol> 7.嘌呤霉素选择针对性的iPS细胞的<ol><li>开始嘌呤霉素选择转染后72小时，当iPS细胞达到70％汇合。 </li><li>对于NCRM5 iPS细胞，开始在0.5微克/毫升嘌呤霉素（1/2全剂量）稀释成NutriStem培养基嘌呤霉素选择。最优嘌呤浓度可能会发生变化，从0.25至1微克/ MLL一些iPSC系。 </li><li>培养iPS细胞在NutriStem + 0.5微克/毫升嘌呤霉素3天，每日刷新介质。 </li><li> 3天之后，增加嘌呤霉素的浓度为1微克/毫升。 </li><li>在1微克/毫升嘌呤选择培养iPSCs的另一个7至8天，或直到明显的菌落出现菌落采摘足够大。 </li></ol> 8.殖民地采摘和扩大目标iPS细胞的<ol><li>利用基底膜基质涂布一个96孔板通过分配50μl的基底膜基质涂层溶液（2毫克基底膜基质稀释到12毫升的DMEM / F12）到每个孔中。存储板在4℃下过夜后使用。 </li><li>使基底膜基质涂覆后的板来室温之后，吸出基底膜基质和分配100μl的E8补充有10μM的Y-27632到每个孔中。 </li><li>放置在倒置显微镜成生物安全柜，并用70％乙醇喷雾轻轻消毒。 </li><li>拉玻璃巴斯德吸管过本生灯，以获得理想的殖民地采摘工具，有一个微小的"钩子"的提示。消毒用70％乙醇，并放置在通风橱中干燥。 </li><li>删除包含从孵化器的目标IPSC的殖民地的菜，并放入显微镜下的引擎盖。 </li><li>通过跟踪菌落周围边框圈与菌落采摘工具挑选iPS细胞。</li><li>使用集落拾取工具的"钩"，以轻轻刮去并从板的表面去除的iPSC集落。对于较大的殖民地，与绘图殖民地采摘工具季度殖民地将有助于重新电镀后的细胞生长的X。 </li><li>一旦细胞团块被分离和自由浮动，使用P200移液器设置为30〜微升收集的iPS细胞。板单元直接放入96孔板中。 </li><li>可选:使用P20移液管设置为〜5微升收集的iPSC成的eppendorf管中，再加入50微升平缓细胞解离试剂，在37℃消化5分钟。消化后，加入500μlE8介质进入eppendorf管以稀释平缓细胞解离试剂和在标准的台式微量离心机降速细胞在200×g离心5分钟。然后吸大部分的培养基中，并离开〜30微升重新悬浮的细胞转移到96孔板中。 注:此做法将促进细胞团块，拿起殖民地的快速扩张的解离。 </li><li>继续菌落采摘直到的iPSC集落的足够数量的已被收集（大约建议每个实验20-30集落）。 </li><li>菌落采摘后，将96孔板在培养箱过夜。刷新次日完整的E8媒体，文化，直到〜70％汇合。 </li><li>传代细胞从96孔到24孔，然后进入6孔，按照步骤4.5-4.10描述。 注:EDTA溶液和E8介质的卷应该使用井比6孔板较小时，可以按比例减少。 </li><li>评估由结PCR和Southern印迹分析靶向成功确认针对性盒一体化随机插入 ​​载体16和不存在。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

对于成功的一代AAVS1安全港的最关键的步骤有针对性的人类iPSCs的是:（1）有效地提供TALEN和捐助质粒成iPS细胞转染; （2）优化的iPSC成单个细胞在转染后的转染和铺板密度之前的解离; （3）优化剂量和基于iPSC系的生长的药物选择的时间; （4）认真剖析，并有针对性的挑选殖民地，转移到新的板/孔。相比于Hockemeyer的纸张10用类似的方法，该协议中使用的一对开源AAVS1-TALENS的，不同的iPSC解离试剂，和不同的转染设备提供TALENS和供体载体，其有助于减少在使用的iPS细胞的数量实验同时实现了高转染和定位效率。 以实现高效率的基因的编辑在人类iPSC，有必要首先优化基因EDI的递送婷试剂（DNA / RNA），同时平衡急性细胞死亡的交付方法和试剂导致iPS细胞。的目标是最大化递送效率，同时保持一个还算低的细胞死亡的程度。因为它是非常容易准备大量AAVS1-TALEN质粒，可以实现与药物的选择在人类iPSC的帮助&gt; 50％HR的效率，这是没有必要从TALEN质粒作出的mRNA。递送的挑战来自大施主质粒，在此情况下是〜10 kb的。它建议使用了AAVS1-CAG-EGFP的供体中的特定人类iPSC系，而不是利用包括在转染试剂盒pMaxGFP测试递送效率，因为pMaxGFP是一个〜3.5kb的小质粒，很容易传递到任何细胞。 AAVS1-CAG-EGFP给体可以使用图1A中所示的限制酶靶向不同的转基因成AAVS1轨迹进行修改。另有12 kb的捐助，其中包含了6.4 kb的录像带，以取代CAG-EGFP，具有可烯成功地用于实现类似的转染和靶向效率（数据未显示）。在一般情况下，人类iPSC其中的难染的细胞类型和如通过流式细胞术检测的GFP +细胞的分析对于大质粒的递送效率可低至5-10％。的转染效率的进一步优化是依赖于人类iPSC成单个细胞的适当的解离，因为细胞团块会降低递送基因编辑试剂到每个小区的机会。该协议采用了温和的和快速的工作细胞分离试剂，但iPS细胞治疗为超过10分钟不建议。通常的iPSC培养小于80％汇合，如果接下来的步骤5.1，使用预温热轻柔细胞解离试剂通常足以以解离的iPSC成单个细胞，轻轻移液保温5分钟。如果不是所有的细胞可以治疗的10分钟之后，将解离为单细胞，因为细胞培养结束-CON流利或殖民地变得非常紧凑，只需收集单细胞从更多的井/板推荐的数量和离开紧紧贴壁细胞后面。重移液不建议，因为机械剪切是更不利的细胞比酶解。当（前通路＃15）与早通道iPSCs的工作，温和解离的做法是对细胞的存活非常重要，因为细胞是已经转染应力比晚通路iPSCs的更加敏感。如果它是难以实现高效率的在两种转染和细胞存活，选​​择给出最高转染效率的做法。转染后，细胞应在将达到50-80％汇合在第3天，当药物选择开始的密度进行电镀。由于每个iPSC系生长方式不同，在转染后铺板密度，可能需要对每个细胞系进行优化。高铺板密度可能导致过度汇合降低DRU的功效克选择，而一个极端低铺板密度可能引起延迟的恢复和药物抗性集落的生长。通常300万起iPS细胞都足以被镀成取决于生存和特定iPS细胞的生长½至210厘米菜肴。同样地，如果使用一个新生成的或者作为-尚未检验iPSC系，产生嘌呤杀曲线建立最低有效剂量在未转染的细胞可以是必要的; iPSC系可以在其选择嘌呤霉素敏感性有很大的不同。该MEF被用于选择，因为他们似乎支持iPSC的增长比在药物选择一些细胞外基质更好。作为一个经验法则，至少5天的嘌呤霉素的1微克/毫升或7天，在0.5微克/需要完成选择毫升。最后，菌落采摘技术是至关重要的保存选药毕竟是有针对性的殖民地。轻柔吹打或分离试剂处理有助于打破殖民地成多块均匀分配到新井，因此可以加快菌落扩张。如果使用分离试剂分解之前拾取殖民地电镀，确保它充分治疗后&gt; 10倍的培养基稀释，因为如果留在一夜之间残余分离试剂能杀死转移的细胞。不管哪种技术被使用，实行殖民地真正的实验前采摘强烈鼓励。 由于所描述的战略利用基因陷阱的方法来推动PAC基因的表达出内源性PPP1R2C子，显著采摘30多个殖民地不应该证明是必要的。在SA-2A联PAC选择在理论上消除了随机整合，只有细胞，但更多的随机整合（S）可发生在iPS细胞成功轴承有针对性的整合。在大多数情况下，几乎100％的药物选择的克隆的有针对性的集成和〜其中10-40％具有附加随机集成公司附件。大部分有针对性的克隆往往有单等位基因目标，虽然实验，其中&gt; 50％的双等位基因靶向确实发生。额外的随机整合的可变频率，以及单相对于双等位基因靶向的比率，是难以控制的。因此，采摘〜20殖民地建议，以确保单或双等位基因靶向只克隆可以得到。鉴于不成功靶向实验中，靶细胞的transfectability，存活和生长速率重新评估，在特定细胞系核酸的功效，以及供体和核酸酶制剂的质量强烈建议之前重新尝试实验的全部内容。 应当指出，类似的基因陷阱施主策略可以用于靶向其它活性的基因，但不适合于沉默基因打靶，需要一个独立的启动子来驱动选择的基因表达。此外，虽然第ËAAVS1安全港被认为具有开放的染色质结构，这并不能保证任何转基因可以强烈地在这个轨迹来表示。事实上，我们最近的一份报告显示，一些弱的推动者无法有针对性的整合在AAVS1座14后，驾驶检测的荧光报告基因的表达。 该协议的重点是使用以及验证TALENS为目标的人类基因组充分研究的安全港轨迹。该技术是高度可再现的和容易适应有任何转基因许多应用。相比于使用随机整合的基因组工程方法，AAVS1-TALEN介导的基因靶向是高度有效的和具体的，在iPS细胞的情况下，结果在有扩大和分化成任何人类细胞类型的潜在稳定荧光细胞。虽然该协议没有描述的设计和设计师核酸验证或有针对性的iPSC菌落p的评估罗珀磁带整合和脱靶分析，一些优秀的协议进行了详细描述了16-18的方法这些方面。使用E8介质无饲养IPSC的文化和传代技术在以前的出版物19也描述细节。上述协议，而对于基因加成入AAVS1安全港人类iPSC的优化，可以作为涉及位点特异性核酸酶介导的基因编辑/使用中的任何细胞类型的同源重组施主除了实验的一般模板。

重新编程人类体细胞分化成胚胎干细胞样诱导多能干细胞（iPS细胞）的能力，首次发现了高桥等人，于2007年1。转导的逆转录病毒表达4转录因子人真皮成纤维细胞（将这样配成山因子的Oct3 / 4，SOX2，c-Myc的和Klf4）被证明是高度相似基于形态学，增殖，基因表达，和表观遗传状态的人类胚胎干细胞（胚胎干细胞）;关键的是，iPS细胞也能分化成所有三种胚层1的细胞。 iPS细胞的增殖潜力和分化的能力，使他们非常有吸引力的工具;通过重新编程从患有特定疾病的患者的细胞，iPS细胞可被用来作为体外疾病模型系统和作为潜在的治疗剂。 对于后者的目的，有几个问题必须的iPSCs的全部潜力之前得到解决在临床环境，可以实现; 在体外培养的人类胚胎干细胞和iPS细胞，重新编程和细胞维持期间使用异种衍生物的致瘤潜力，并且需要在体内跟踪移植细胞都是至关重要的障碍的临床应用的多能干细胞的（来自Hentze 等人 。 2）。一个理想的解决方案的需要跟踪分化的细胞移植后会涉及目视可检测的标记物抵抗沉默和花斑不管应用程序的。综合转基因稳健和持续的表达是最容易实现的，当外源DNA导入安全港位点;即，基因组位点，使综合向量的足够的转录，而在同一时间减轻表达扰动邻近基因3。一个这样的网站已经非常好特点，因为它的发现是腺相关病毒INTEGRATION站点1（AAVS1），在蛋白磷酸酶1调节亚基12C（PPP1R12C）基因的第一内含子。该位点已被证明不仅以允许持续和通过延长时间鲁棒表达的整合的转基因在培养和体外分化3，但也保护周围的基因从转录扰动4;两个特征被认为是由于内源染色质绝缘体元件侧翼AAVS1部位5的存在。 在刚刚过去的十年进展基因工程工具，极大地促进了易用性和效率与遗传操作的任何细胞类型可以实现的。虽然早期的成功实验，依靠内源性同源重组（HR）的极低水平与引进捐助来实现基因在胚胎干细胞6,7目标，采用特定地点核酸酶，如锌指核酸酶（ZFN），即显通过双链DNA断裂的产生ficantly诱导同源重组大大增加了这样的实验8,9的效率。这两个转录激活因子样效应植物病原黄单胞属（故事）和原核聚集定期相互间隔短回文重复（CRISPR）/ Cas9系统进入高效的站点特定的设计师核酸酶的再利用已经取得了基因打靶的多能干细胞的访问，可行的方法10-13。 最近的一篇论文描述了用于稳定整合绿色荧光报道基因盒的成在使用TALE核酸（TALENS）14人类iPSC的AAVS1安全港轨迹的有效方法。这些有针对性的iPS细胞保持其荧光即使在定向分化为心肌细胞，并移植到心肌梗死（MI）的小鼠模型，提供了强有力的证据，这样的效用稳定地荧光多能干细胞14。以获得目标菌落，基因陷阱方法用于其中一个拼接-受体（SA），2A自我裂解肽序列置于内源性PPP1R12C启动子的控制下的嘌呤霉素N-乙酰基转移酶（PAC）的基因;因此，只有那些掺入该DNA供体的AAVS1轨迹的iPSCs表达PAC，使它们可选择基于嘌呤霉素抗性; （ 图1，15）。该协议的细节产生AAVS1-GFP iPSCs的报道，在最近的一篇文章14，包括iPS细胞转染与TALENS的过程和9.8 kb的捐助为4.2kb DNA片段插入AAVS1安全港轨迹整合，选择iPS细胞的基础上，程序嘌呤霉素抗性，和用于克隆扩增拾取菌落。本文描述的技术可以应用到许多基因组工程实验。

<table><tbody><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml</td><td>Corning</td><td>354230</td><td>Store at -20 &amp;deg;C.</td></tr><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>11320-033</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Essential 8 Medium</td><td>Life Technologies</td><td>A1517001</td><td>Store basal medium at 4 &amp;deg;C. Store supplement at -20 &amp;deg;C.</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td>Store at room temp. Once dissolved in H&lt;sub&gt;2&lt;/sub&gt;O, store at -20 &amp;deg;C.</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S5886-500G</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Life Technologies</td><td>14190-250</td><td></td></tr><tr><td>100 mm TC-Treated Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>DR4 MEF 2M IRR - Academic</td><td>GlobalStem</td><td>GSC-6204G</td><td>Store in liquid Nitrogen.</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995-040</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Defined Fetal Bovine Serum, US Origin</td><td>HyClone</td><td>SH30070.03</td><td>Store at -20 &amp;deg;C. Thaw at 4 &amp;deg;C overnight and aliquot. Store aliquots at -20 &amp;deg;C until needed.</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Life Technologies</td><td>11140-050</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>4D-Nucleofector Core unit&amp;nbsp;</td><td>Lonza</td><td>AAF-1001B</td><td>part of the electroporation system</td></tr><tr><td>4D-Nucleofector X unit&amp;nbsp;</td><td>Lonza</td><td>AAF-1001X</td><td>part of the electroporation system</td></tr><tr><td>P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)</td><td>Lonza</td><td>V4XP-3024</td><td>Upon arrival, remove Primary Cell Solution and supplement and store at 4 &amp;deg;C.</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Life Technologies</td><td>A1110501</td><td>Store at -20 &amp;deg;C. Thaw overnight at 4&amp;deg;C and warm an aliquot in a 37 &amp;deg;C water bath before use.</td></tr><tr><td>NutriStem XF/FF Culture Medium</td><td>Stemgent</td><td>01-0005</td><td>Store at -20 &amp;deg;C. Thaw overnight at 4 &amp;deg;C</td></tr><tr><td>AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td><td>Addgene</td><td>52637 and 52638</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>AAVS1-CAG-EGFP Homologous Recombination donor</td><td>Addgene</td><td>22212</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>Life Technologies</td><td>A11138-03</td><td>Store at -20 &amp;deg;C. Prepare working aliquots of 1 mg/ml in ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-20A</td><td></td></tr><tr><td>Sorvall Legend XTR (Refrigerated), 120 V 60 Hz</td><td>Thermo Scientific</td><td>75-004-521</td><td></td></tr><tr><td>TX-750 4 &amp;times; 750 ml Swinging Bucket Rotor</td><td>Thermo Scientific</td><td>75003607</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr></tbody></table>

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.

该协议的可视化图2提供的，用句其间iPS细胞在不同介质中无论是绿色E8或蓝色的NutriStem突出培养。它转染仅高质量iPSCs的是很重要的;检查培养皿整个例行维护和验证的iPSC培养包含主要鲜明菌落轴承鹅卵石样形态（ 图3A）;分化的细胞不应该占据的培养物的10％以上。 iPS细胞的Transfectability是由于它的小尺寸的评估和优化使用小PMAX-GFP载体（ 图4A-B）中 ，如转染PMAX-GFP通常表示可达到的最大效率。例如，我们与PMAX-GFP（ 图4B）达到68.6％的转染效率。瞄准AAVS1安全港，iPS细胞使用的是温和的单细胞分离试剂传代，并转AAVS1-TALENS和AAVS1-CAG-EGFP（在图1A中所描绘的质粒）。转染的iPSC然后铺于DR4的MEF（图3B）的适当密度。的AAVS1-CAG-EGFP的峰在48到72小时后转染（ 图3C）的瞬时表达;如果需要的话，转染的iPS细胞的一小部分可以被FACS分析。 NCRM5 iPSCs的可转用AAVS1-CAG-EGFP的供体（ 图4C）60.9％的效率。转染效率可以相差很大;对于实验，其中的GFP +级分甚至低至10％，继续嘌呤选择。进行嘌呤霉素选择后，单个克隆显示均匀的荧光应该对菌落拾取（ 图3D）足够大。挑克隆，一个合适的菌落位于下低倍率（ 图3E），并且由第一跟踪分离和营它（ 图3F-G）。在驻扎菌落然后轻轻刮去d从培养容器（ 图3H）。用P200移液器，细胞团块，然后转移到基底膜基质包被的96孔平板的一个孔中。 iPS细胞应2-4小时电镀（ 图3I）内安装。如果附件未在24小时内观察到的，基底膜基质涂层或Y-27632治疗很可能亚最佳;基底膜基质涂层新的96孔板和采摘更多的殖民地之前准备新鲜的E8 + 10μMY-27632。一旦在96孔格式中，有针对性的iPSC克隆扩充，并应表现出的绿色荧光蛋白（ 图4D-E）的稳定和均匀的表达。 <img alt="图1" src="/files/ftp_upload/52504/52504fig1highres.jpg" /> 图1:基因打靶载体和定位原理。在AAVS1-CAG-EGFP的（A）陈述，以及PZT-AAVS1-L1 / R1 TALENS质粒在这项研究中使用。该SA-在AAVS1-CAG-EGFP质粒2A元素表示用于限制嘌呤霉素N-乙酰基转移酶（PAC）的基因表达的靶向整合事件的拼接 - 受体和2A自我裂解肽。鸡β肌动蛋白球蛋白（CAG）的启动子用来驱动的EGFP的表达。（B）中的AAVS1安全港，包含在PPP1R12C基因的内含子1，是使用TALENS以产生双链DNA断裂的针对性。这将激活同源重组（HR）修机械，其然后使用AAVS1-CAG-EGFP的供体（轴承同源臂侧翼切割位点）作为底物进行修复。磁带盒被整合和PAC基因置于内源性PPP1R12C启动子的控制之下。 <img alt="图2" src="/files/ftp_upload/52504/52504fig2highres.jpg" /> 图2，基因打靶实验时间轴。iPS细胞是培养至70％汇合，并传代〜1:6 -2天的（d-2）。的MEF铺板在d-1，和iPSCs的收集和转染在D0。在D1，媒体被切换到NutriStem和iPS细胞对于两天培养之前嘌呤加入到D3中的介质。菌落一般准备由D12采摘。时段中iPS细胞是E8培养基突出显示绿色，而NutriStem文化时期是蓝色的。 <img alt="图3" src="/files/ftp_upload/52504/52504fig3highres.jpg" /> 图3:iPSC的基因打靶代表图像的高品质的iPS细胞（A）相图像。注意相亮边界和鹅卵石样形态（B）的 DR4的MEF铺板于2×10 4个细胞/ cm解冻后224小时。（℃）72小时后转染，EGFP +细胞是显而易见在荧光MICR看时oscope需要。（D）的后嘌呤为基础的选择为〜14天，菌落显示均匀GFP荧光大到足以被拾取。（EH）菌落拾取过程的代表性图像。菌落用挑一拉巴斯德吸管中，首先通过概述的菌落，然后用四分法它以获得更小的细胞团块。（I）中精选的iPSC集落附着于96孔板的基底膜基质涂覆的表面在2至4小时。 <a href="https://www.jove.com/files/ftp_upload/52504/52504fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52504/52504fig4highres.jpg" /> 图4:转染效率和克隆靶向iPSCs的FACS分析。 （A）控制NCRM5 iPS细胞没有任何质粒转染。（B）NCRM5iPS细胞转染了小试验载体PMAX-GFP是FACS分析GFP的表达。（C）中AAVS1-CAG-EGFP质粒的瞬时表达通过FACS评估。 （D）的NCRM5-AAVS1-CAG-EGFP克隆显示器作为检测用流式细胞仪一致正EGFP表达。注意分析iPS细胞的紧张集群相比，C）。（扩大NCRM5-AAVS1-CAG-EGFP针对性克隆E）荧光图像。

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

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

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 ...

transfection-selection-colony-picking-human-induced-pluripotent-stem

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

19.6K 次观看.  National Institutes of Health. 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.

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

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

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

癌症研究

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

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

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

神经科学

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

细胞培养物中的粗载体生产和高效 DNA 递送

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

作者聚焦:揭示未纯化重组 AAV 在细胞培养研究中的潜力 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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

摘要

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此视频中的章节

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

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

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

人类诱导多能干细胞的转染和Nucleofecting

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

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

基于增强子的表达构建体在小鼠大脑中的 AAV部署

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达

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

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在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

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

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

人类诱导多能干细胞的转染和Nucleofecting

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

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达

基于增强子的表达构建体在小鼠大脑中的 AAV部署