T.R.R conceived the project, designed and performed experiments and prepared the manuscript. E.J.K and S.E.M performed experiments. All authors contributed intellectually to the final manuscript. This work was funded by a University of Leicester Innovation Fellowship to SEM and by a UK Medical Research Council (MRC) Senior Research Fellowship to S.M.C (MR/J009202/1). DNA sequencing was performed by Protein and Nucleic Acid Laboratories (PNACL), Centre for Core Biotechnology Services, University of Leicester. We wish to thank A. Francis Stewart for providing the Rox and Dre plasmids. pL451 and pL452 plasmids were obtained from National Cancer Institute (NCI), Frederick, USA.

1.基因打靶设计<ol style="margin-left: 40px;"><li>订购合适的BAC克隆覆盖感兴趣的基因组区域。确保这是等基因对ES细胞的类型来修改例如 RPCI-23和RPCI-24 BAC克隆用于基因靶向与C57BL / 6 ES细胞。 </li><li>应用传统设计基因的靶向载体时的定位标准。关键参数包括修改是否需要为组成型或有条件的，限定在内含子盒的一个基因敲除策略和间距和安置临界外显子（CE）为删除。 注:所有这些已作了详细讨论别处10。 </li><li>选择的基因组区域，每个区域的5-6 kb的侧翼目标修饰位点。 注:因此亚克隆插入片段的大小通常是10-12 kb的，虽然上限可高达80 kb的具有低拷贝亚克隆像P15A质粒pBR322的等，并且高达200 kb的与BAC向量。 </li></ol> 2.多重重组工程寡核苷酸<ol style="margin-left: 40px;"><li>设计寡核苷酸用于质粒骨架（亚克隆质粒）和选择性标记（插入磁带盒）。设计每个寡聚使得它含有180 bp的HA侧翼的基因组的目标位点和20碱基的插入盒或亚克隆质粒（ 图2）的具体引发序列。 注:同源臂不应包含任何重复的元素，重复的元素的存在将导致不正确的定位和亚克隆。重复元件可以通过使用基于网络的工具，如&#39;重复掩蔽&#39;&#39;被检测到。 </li><li>包括一个独特的限制性内切酶（RE）位点上的矢量寡聚之一来线性为ES细胞靶向（ 图2）的基因打靶载体。 </li><li>检查使用寡分析程序寡参数。小心避免在引发区二级结构秒。 </li><li>较短的HA（50碱基对）的插入盒的被容许的，产生的重组体的数量较少，但确保了亚克隆质粒的HA总是较长（180碱基对）。 </li><li>确定使用诸如&#39;&#39;cloneDB&#39;基于网络的工具的特定BAC克隆的基因组DNA插入物的取向。通过检查BAC文库构建中使用的BAC质粒骨架的地图建立从ORIS复制的方向。 注:ORIS通常是相反的氯霉素（叶绿素）标记为所有常用的BAC质粒的转录方向。 </li><li>添加两个终端硫代磷酸酯（PTO）键合到寡核苷酸的相反复制的BAC克隆的方向的5&#39;端。添加5&#39;磷酸修饰的反向寡（ 图2）。 注:非对称S代PCR录像带时红消化产生的单链DNA中间体，可贷的落后股的复制叉。 注:最大多路复用重组频率观察到与后随链的保护盒。前导链保护的，或在某个位点双重保护的磁带可能不会产生相同的重组效率。 </li><li>订购寡核苷酸与PAGE或HPLC纯化。 </li></ol> 3. BAC克隆转型与pSC101 BADgbaA重组工程质粒。 <ol style="margin-left: 40px;"><li>使大肠杆菌大肠杆菌菌株轴承的BAC重组工程精通，转换它与含有λRed基因16的pSC101 BADgbaA质粒。 注:pSC101 BADgbaA质粒含有阿拉伯糖诱导型阿糖胞苷-P BAD启动子，四环素选择标记和所述温度敏感pSC101复制子的控制下的红色和的RecA基因。 <ol><li>刺无菌枪头在BAC琼脂培养基，接种5.0毫升LB培养基中（LB），含12.5 pH值8.056克毫升-1叶绿素。生长于37℃5小时以200rpm振荡。 </li><li>寒意10％（ 体积/体积 ）甘油溶液，微量离心管，并在冰上电试管。冷却冷冻离心机大型离心和4℃。 </li><li>确定使用分光光度计培养物的光密度（OD），测量600nm处吸光度。制备电感受态细胞（如下文所述），当达到的OD 600读数为0.3至0.8。 </li><li>降速细胞在50毫升离心管中的大离心机在1216×g离心5分钟，在4℃。 </li><li>用1ml冷却的10％甘油洗涤细胞和降速细胞在17949×g离心20秒，在4℃。执行洗涤，共3次的步骤。 </li><li>将细胞重悬于50微升10％的甘油的总体积，并添加10-200纳克pSC101 BADgbaA重组工程质粒。得到单细胞悬浮液通过移上下数次，然后将细胞转移到预冷1毫米间隙电穿孔杯中。 </li><li>电穿孔的细胞的1.8千伏，25μF和200Ω的设置。 注:请正确的设置每个品牌电穿孔的。电穿孔的时间常数小于4表示的盐和其它杂质的存在。 </li><li>立即恢复细胞在1ml的LB，将细胞转移到50毫升离心管中。 </li><li>生长上述BAC gbaA培养在30℃下2小时以200rpm振荡。 注:当细胞生长在37℃，由于温度敏感REPE复制因子的失活的pSC101 BADgbaA质粒丢失。生长gbaA细胞在30℃下维持重组工程功能。 </li><li>添加9毫升LB含12.5微克毫升-1叶绿素和4微克毫升-1四环素（TET），以回收的BAC gbaA文化。成长BAC gbaA文化O / N为30 &#160; ℃，200rpm振荡。 注:电穿孔超螺旋gbaA质粒的转化效率是足够高的，以允许饱和生长的O / N的液体介质。 </li></ol></li></ol> 4.准备插入纸盒和亚克隆质粒<ol style="margin-left: 40px;"><li>使用长的寡核苷酸如下所述合并的HA到插入盒（S）和亚克隆质粒，进行聚合酶链反应（PCR）。 <ol><li>可选:为防止交叉污染质粒进入重组工程反应，使用R6K出身或类似窄宿主范围质粒模板扩增插入磁带。 </li><li>可替代地，使用线性化的RE消化物（ 表2）的质粒模板。选择一个RE，削减PCR扩增区域外，是热灭活。热灭活的RE所推荐的MANUFacturer。 </li><li>建立使用高保真热启动DNA聚合酶系统聚合酶链反应（PCR）。准备PCR预混详见（ 表3）。所示（ 表3）进行热循环。 </li></ol></li><li>分析PCR产物通过琼脂糖凝胶电泳。负荷1-5微升每个PCR的于1％（重量/体积）含有0.5毫克毫升-1溴化乙锭（EtBr的）的琼脂糖凝胶。 注:非特异性扩增产物的存在不会在重组工程干扰反应。在某些情况下，然而，引物二聚体可能降低重组工程效率。 </li><li>纯化使用PCR纯化试剂盒的PCR产物。 <ol><li>可选:通过DPNI处理后经PCR清理删除的PCR质粒模板。在无菌去离子水的最小体积洗脱的DNA（如制造商推荐的）。 注:加DPNI来未纯化PCR反应结果届裂解的效率下降Ë甲基化的质粒DNA模板。 </li></ol></li><li>量化通过琼脂糖凝胶分析PCR扩增的DNA对一组已知的DNA标准品例如 ，λ的HindIII消化或通过使用NanoDrop分光光度计。 </li></ol> 5.亚克隆插入加<ol style="margin-left: 40px;"><li>通过在10毫升LB +叶绿素+春节增加200微升稀释O / N BAC gbaA文化的50倍。增长30 &#160; ℃，200rpm振荡1小时50分钟。包括样品用作阴性对照。 </li><li>寒意所有重组工程的材料和设备，以4℃，如在步骤3.1.2说明。 </li><li>倾LB琼脂的pH含有适当抗生素的选择性（S），将允许选择被插入以及用于亚克隆载体（ 表4）的选择的DNA片段的正确的浓度为8片。 注:某些抗生素的pH敏感。使用LB琼脂pH值为8的规则。 </li><li>制备10％（重量/体积）L-阿拉伯糖的溶液。过滤消毒用0.2μm注射器过滤。 注:阿拉伯糖诱导从gbaA质粒的重组工程蛋白质的表达。 </li><li>检查使用分光光度计的BAC gbaA文化的OD。一旦OD达到600 0.25-0.3，如在以下步骤中描述诱导的重组工程的蛋白质。 </li><li>加入200微升10％的阿拉伯糖溶液至10毫升的BAC培养物达到0.2％阿拉伯糖的终浓度。包括未诱导培养（不含阿拉伯糖）被用作阴性对照。 </li><li>转移BAC文化37 &#160; °下振荡培养箱，诱导表达红色45分钟晃动在230转。 注:红蛋白的表达是​​低效的，在30℃。 </li><li>降速细胞和如在步骤3.1.5中所述用10％甘油的3倍。 </li><li>添加600-1，000纳克每一个亚克隆质粒和插入卡带（S）的重组工程反应。只包括一个矢量和矢量加上单个插入控件来检查载体和盒的重组熟练性和完整性。 </li><li>通过上下吹打获得单细胞悬浮液。如在1ml的LB，在步骤3.1.7和随后的恢复描述在37℃下进行1小时的多拷贝质粒或在10毫升的LB中于37℃下进行3小时，为的BAC载体进行电穿孔。 </li><li>将回收的培养例如，90％，10％，在双重选择琼脂平板1％和增长37的板不同稀释&#160; °下16小时。 </li></ol>重组子6.分析<ol style="margin-left: 40px;"><li>可选:选择6〜12殖民地和进行菌落PCR使用HA侧翼引物和插入特异性引物。包括亚克隆质粒和插入CAS塞特质粒以及父BAC克隆作为阴性对照。 <ol><li>执行菌落PCR的琼脂糖凝胶分析。由亮带的预期大小存在确定阳性克隆。 注:SPI克隆过程的高效率产生大多是正确的重组。 </li></ol></li><li>挑克隆到5ml的LB pH值为8包含选择性抗生素和成长O / N在37℃。 </li><li>准备使用柱纯化试剂盒按照制造商的说明DNA的小量制备。 </li><li>使用标准的苯酚 - 氯仿分离或类似的协议准备的BAC小量制备的DNA。 </li><li>在小量DNA进行RE消化。分离的DNA通过琼脂糖凝胶电泳。分析的RE型态以鉴定含有正确定向向量的预期片段大小的克隆。选择的RE的载体缺少插入件（S）和含有插入物（S）的向量之间清楚地辨别。 </li><li>宾得纳尔:获取单元，缺少的BAC，它仍然存在于大肠杆菌的大肠杆菌重组工程后，用小量质粒DNA转化DH5alpha或DH10B E.大肠杆菌细胞。 </li><li>进行DNA测序通过HA和插入磁带检查寡核苷酸合成的错误。 </li></ol>

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</ol>

T.R.R is the inventor of a British patent application on multiplex recombineering technology.

胚胎干细胞系和小鼠模型建设历来参与基因使用包含修改后的等位基因4质粒构建目标。然而，这些复杂的基因打靶载体的结构已被证明是在及时制作这种模型的一个显著瓶颈。基于重组工程载体的构建发展战略，使得改进的矢量设计和更有效的载体组装。尽管如此，目前的重组工程的协议仍然涉及多个步骤，需要中间质粒纯化，并使用不同的菌株。亚克隆加上插入提供了一个新途径，可以在一个电活动在驻地BAC宿主菌进行载体构建。 SPI的基因打靶的效用此处进行试验的各种载体的构建的应用程序。在这里检测的所有实例中，SPI被证明是有效的和正确的重组质粒制备。在大多数的情况下，多个不同的盒被正确插入在目标载体。大DNA盒和载体中容易地容纳在SPI协议和显示了该系统的灵活性。 SPI依靠使用长的同源序列和硫代磷酸酯（PTO）保护的线性DNA盒的。 PTO修饰赋予保护，防止核酸外切酶的线性DNA 20,21和长的HA增加复合效率，以允许多路复用。然而，合成的长寡聚序列增加累积误差特别缺失的可能性。如果它们包括蛋白编码区的寡核苷酸突变可能是特别有害的。通过HA DNA测序和覆盖所述插盒的全长强烈建议，以消除任何序列改变的克隆。采用高保真DNA聚合酶系统还建议，以避免引进的ŸPCR错误。亚克隆质粒的HA的长度和组合物是更关键的相对于所述插入磁带盒的（数据未示出）。对于特别敏感的应用程序，如建设敲入载体，其中外显子区域的任何突变不会容忍的，插入卡带的HA可被缩短（50-120基点），以避免长时间的寡核苷酸相关的问题。插入暗盒，也可以保持不变或双硫化磷酸酯化（其中，复制方向的知识是不可用）。但复用在这些情况下，仍需要很长的保护HA亚克隆质粒。这个特殊的战略的一个需要注意的是复用效率，可以潜在地影响SPI克隆在不同位点的降低。 插入盒和亚克隆质粒的长度是在SPI克隆的另一个重要参数。较大的DNA分子的电穿孔效率较低，效果是累积的，给定的重quirement引入在同一小区内的所有盒（数据未示出）。复用是最有效的较小插入磁带。 DNA片段大于3 KB也放置限制在Red介导的单链DNA的处理，这是最有效的达到3千30。插入磁带超过3 kb的是双切除，并通过独立的测试通道20重组效率较低。因此，具有足够的菌落，以确定正确的克隆筛选成为在这些情况下，非常重要的。重组后的电持续时间较长也增加了困难SPI练习正确的克隆复苏的机会。多达四个小磁带盒（&lt;1.5 kb的）可以被同时插入与SPI过程中，虽然与每个附加盒的复用效率降低（数据未显示）。然而，这反映了一个最佳的SPI实验的结果，并最好是用许多大型磁带盒时要考虑的多路复用的极限。 </P&gt; 的基因组编辑工具的发展像群集定期相互间隔短回文重复序列（CRISPR）-cas9内切酶系统使建立新的基因组修饰，并导致更高效的基因组工程36。然而，这些新技术已经补充基因靶向载体，而不是取代他们。据设想，该CRISPR-CAS系统可以代替抗生素选择和进一步缩小多路复用重组工程协议。

基因打靶技术的发展已使细胞系和小鼠模型的结构，研究了不同的生物系统1-3。在一个基因组序列的修饰的关键第一阶段是靶向载体4的基因的设计和施工。靶向载体质粒构建携带感兴趣含有选择标记（ 例如，新霉素 ）两侧所需的修改（多个）的等位基因，和在哺乳动物细胞中5需要有效的同源重组长的基因组区域。精确的基因修饰被引入靶向载体导入胚胎干细胞（ES细胞）或6的体细胞7，实现由此在靶载体和DNA序列的相同的延伸之间的同源重组的基因组基因座的结果在期望的修饰的转移通过基因转变8的基因组中。这种modifi编ES细胞可以被注射入小鼠胚泡以产生后代（嵌合体），那么，可以通过与种系9发送这些改性等位基因。备用路由，以产生转基因小鼠包含一个基因表达载体导入单细胞小鼠受精卵，这导致了随机整合在小鼠基因组10中的载体的显微注射。传统载体构建的方法已依赖于常规"剪切和粘贴"使用限制性内切酶和DNA连接酶来克隆不同选择标记和基因组片段插入载体主干克隆。然而，传统的克隆的固有的限制因素是定位和选择限制性位点，尤其是具有较长的DNA序列。这通常需要多个亚克隆步骤，还引入了外源DNA序列插入载体，这往往会导致基因的较低效率定位。 重组工程（重组的ENGineering）是一种DNA工程技术，通过使用由在大肠杆菌噬菌体的重组蛋白介导的同源重组（HR）克服了这些限制大肠杆菌细胞11,12。因为同源序列的任何区域可作为重组工程的基板中，限制位点的可用性的限制被去除。大的DNA序列可以被无缝地在体内直接修改，因此也保持它们的结构完整性13。重组工程是非常有效的短同源性（50碱基对）14，因此，同源臂（HA）可以方便地合并到合成寡序列。在一个典型的重组工程实验，寡或含HA的双链DNA（dsDNA）的片段被电穿孔入重组工程感受态E.的大肠杆菌细胞含有位于无论是在染色体或在质粒上15的靶。重组电位由红REC的诱导型表达赋予ombination蛋白质噬菌体16,17的或外消旋噬菌体18的RecET蛋白质。红/外切酶的RecE线性双链DNA转化到单链DNA（ssDNA）的中间，然后由它的伙伴，红/ RECT，单链退火蛋白（SSAP）19的约束。长的ssDNA或短寡与其互补靶序列退火发生在复制叉的后随链，并导致在靶位点的序列的掺入。随链的ssDNA重组是重组工程的高效率的基础上，并可以由"测试"重组模型20,21进行说明。 一个典型的重组工程的工作流程，以建立一个基因靶向载体包括以下两种路线。一个途径涉及亚克隆从小鼠BAC克隆所需基因组区到质粒随后顺序插入的loxP重组位点，选择标记<s向上&gt; 22等，或替代的路线涉及靶向的BAC基因组基因座与由多轮重组工程10,23，然后亚克隆改性轨迹到质粒通过缺口修复克隆24,25的不同目标矢量元素。有关这一主题的变化已被用于在不同的高通量重组工程管道作为大小鼠生产方案26,27的一部分。然而，这些程序涉及复杂和冗长的阶段，需要使用专门的载体和大肠杆菌大肠杆菌菌株（ 例如，Cre表达的细胞），并利用一个或多个中间步骤载体DNA纯化和再转化（ 表1）。亚克隆插入加（SPI），是一种将β重组和缺口修复克隆到一个单一的过程（ 图1）的新型重组工程技术。 SPI向量组装简单，快捷，灵活，并提供标准recombi显著改善neering接近（ 表1）。在这里，我们展示了易用性和使用SPI与特别强调构建非标准和具有挑战性的矢量设计不同载体构建的应用程序。试验例包括荧​​光报道敲入载体的结构中，双标记的蛋白表达敲入载体，BAC荧光报道载体和条件性敲除载体。这些研究各种要求本地化的细胞表面受体，纯化核蛋白质复合体或有条件地烧蚀的基因的表达。

<table><tbody><tr><td>Antibiotics except Zeocin</td><td>Sigma: http://www.sigmaaldrich.com</td><td>Ampicillin, A9518-5G;&lt;br/&gt; Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G;&lt;br/&gt; Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td><td></td></tr><tr><td>Zeocin</td><td>Invivogen:&amp;nbsp;</td><td>ant-zn-1</td><td>Zeocin is also available from Life technologies/Fisher</td></tr><tr><td>C57BL/6 BAC clones</td><td>CHORI: https://bacpac.chori.org/&amp;nbsp;</td><td>RPCI-23 or RPCI-24 BAC libraries</td><td>129/Sv BAC clones can be obtained from Invivogen or Life technologies/Fisher</td></tr><tr><td>KOD hotstart DNA polymerase system</td><td>Millipore: https://www.merckmillipore.com</td><td>71086-3</td><td></td></tr><tr><td>L-Arabinose</td><td>Sigma: http://www.sigmaaldrich.com</td><td>A3256-25G</td><td></td></tr><tr><td>Long oligos</td><td>IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td><td>IDT Ultramers or Gene Link long oligos</td><td>PTO and Phosphosphate available as custom modifications. PAGE purification strongly recommended for long oligos.</td></tr><tr><td>MinElute PCR purification kit</td><td>Qiagen: http://www.qiagen.com&amp;nbsp;</td><td>28004</td><td>Minimum elution PCR purifications kits are preferred.</td></tr><tr><td>QIAPrep Spin Mini Prep kit</td><td>Qiagen: http://www.qiagen.com&amp;nbsp;</td><td>27104</td><td>Silica column or similar matrix kits are preferred.</td></tr><tr><td>Restriction enzymes</td><td>NEB: https://www.neb.com</td><td>DpnI: R0176L</td><td>See NEB website for a list of RE.</td></tr></tbody></table>

subcloning plus insertion (spi) - a novel recombineering method for the rapid construction of gene targeting vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

敲入矢量 敲入打靶载体反映需要引入一个新的序列特征中，包括单碱基对的取代在一个蛋白编码区，一个荧光标记物的融合或亲和标记的蛋白质或基因表达盒的整合的基因组。为了测试SPI的敲入载体的构建策略的应用，两种不同的测试案例进行了研究。Dnttip1编码脱氧核苷酸转移酶，终端，相互作用蛋白1A（TDIF1）与类聚我去乙酰化酶（HDAC）形成有丝分裂脱乙酰基酶复合体（MIDAC） 28。调查Dnttip1在细胞分裂中的作用，串联亲和标记方法被送往隔离TDIF1相互作用的蛋白质。以前的尝试使用含有长同源区域一个P15A载体亚克隆Dnttip1基因的一部分导致低缺口修复效率次频繁异常重组产物（数据未示出）。因此，在Dnttip1轨迹的敲入载体的结构提供了一个具有挑战性的重组工程工作。甲SPI策略旨在亚克隆Dnttip1基因跨越最后的外显子（外显子13）的12 kb的部分为低拷贝P15A矢量，并同时插入一个双亲和标签选择盒插入的外显子13，取代了终止密码子（ 图3A ）。该2X FLAG-钙调蛋白结合蛋白（CBP）联系FRT-PGK-EM7，新霉素（NEO）-BGhpA-FRT卡带（2.0 KB）从使用包含120 bp的HA侧翼Dnttip1双PTO修饰的寡核苷酸线性质粒的RE放大终止密码子。甲P15A 沸石Dnttip1亚克隆载体（1.7kb的）构建含有200bp的区域同源的序列Dnttip1的端部到进行亚克隆。该Dnttip1亚克隆质粒重新线性化，用20碱基修饰的寡核苷酸PCR扩增生成一个前导链保护的载体。将PCR产物进行DPNI处理，提纯，共电穿孔进入重组工程主管Dnttip1 BAC E.大肠杆菌细胞以及未诱导的对照细胞。 SPI的反应物铺板于吉欧霉素（Zeo的）和卡那霉素（KAN）含有琼脂板（ 图3B）。 SPI的产生在所有的12个重组体进行分析（ 图3C）的正确修改Dnttip1敲入载体。 DNA测序证实的情况下，从寡核苷酸合成或PCR扩增产生的任何错误。 SPI的另一例所涉及的在帧插入一个增强型黄色荧光蛋白（EYFP）的链接的选择盒在P2rx1基因。所述P2rx1基因编码G蛋白偶联受体，其功能如同一个ATP门控离子通道29。联动YFP的荧光允许在VA细胞表面上的P2X1受体的跟踪rious功能测定。P2rx1是已经提出显著问题与常规重组工程方法的另一个困难轨迹（数据未示出）。采用SPI，所述P2rx1基因包含所述终端的外显子（外显子12）的12 kb的片段被亚克隆到P15A 沸石载体和改性的EYFP -LoxP侧翼Neo盒替换的终止密码子的外显子在12来构造的协同插入P2rx1-EYFP敲入载体（ 图3D）。在这个例子中，滞后链保护的P15A载体（1.7 kb的）含有230 bp的HA和EYFP磁带包含50个基点HA和也保持不变。该EYFP插入卡带（2.7 KB）组装拼接重叠PCR的EYFP基因，PCR从pEYFP-C1放大，而LoxP- PGK-EM7-NEO-BGhpA-LoxP位卡带，PCR从pL452（NCI，弗雷德里克）放大。 SPI的反应产生数百菌落（数据未示出）。 RE DNA分析从11个克隆制备小量制备表明大部分包含在正确组装P2rx1-EYFP敲入载体（ 图3E）。通过DNA测序附加验证表明无差错的EYFP盒插入。然而，一些克隆显示出的未标记缺口修复，在相同的细胞（泳道6-9）的标记的缺口修复质粒轮廓。一些样品中还含有不正确缺口修复（2道）或错误定位质粒（泳道4和5）。使用大磁带盒（&gt; 3 KB），从约束长的DNA片段30的红色合成能力，或者如果使用短的HA其出现时并发亚克隆并在某些SPI重组体靶向的失败突出高效的SPI克隆的限制。实际上，增加了EYFP盒200碱基的HA增加的SPI效率和磁带盒的正确定位（数据未示出）。基因与P2rx1目标-在JM8.N4 EYFP敲入载体小鼠胚胎干细胞（C57BL / 6株）产生的6个阳性克隆出96个，其中载有正确的目标P2rx1-EYFP序列（ 图3F）。 BAC报告载体 靶基因的BAC克隆常常包含所有必要的上游和下游调控元件例如增强子，非编码区等，以及内源性启动子来驱动基因表达在天然水平31。因此，一个BAC报告载体是首选的车辆复述的基因32的内源性表达模式。然而，BAC质粒（高达200 KB）的大尺寸呈现显著实际问题转一个完整的BAC进入细胞33。通过BAC修整34,35，同时保留的基因的必需调控元件减小的BAC基因组插入物的大小，能够更容易地处理的BAC和结果以更有效的transfecti上。目前BAC工程技术涉及到多轮重组工程，以实现这一目标35。为了演示的SPI的效用在BAC修剪，一个pBeloBAC11 BAC载体被用于亚克隆30 kb的基因组序列，包括来自168 kb的P2rx1 BAC全长P2rx1基因连同EYFP盒中P2rx1基因同时插入（ 图4A）。含180 bp的HA的pBeloBAC11 沸石载体骨架（6.5 KB）的PCR扩增生成一个滞后链保护的载体修饰的寡核苷酸。同一EYFP Neo盒（3 KB），在之前的P2rx1敲入载体的构建用PCR扩增使用含有180 bp的HA随链保护寡核苷酸和有针对性的P2rx1外显子12代替终止密码子。继EYFP盒综合电和pBeloBAC11 ZEO载体导入P2rx1 BAC细胞EXPR咝声的gbaA重组工程蛋白质，所述培养物中回收在10毫升的LB pH为8 3小时在37℃下，以分隔所述BAC质粒。纯重组体上Zeo的和Kan选择琼脂平板，并在2×10 -6的频率进行观察。菌落PCR的基因分型分析表明成功的BAC修整在3出6个克隆被分析（ 图4B）。横跨EYFP插入位长范围PCR扩增证实了正确的盒整合到3个阳性克隆（ 图4B）。三个克隆缺少EYFP刀片也错误地间隙修复在5&#39;端，尽管EYFP盒在这些克隆误炸的原因可能是单独的5的正确闭合"的BAC末端。三个EYFP阳性BAC克隆用的RE消化物进一步分析表明，正确地修整EYFP重组的BAC（ 图4C &lt;预期图案/ STRONG&gt;）。序列分析显示不存在在EYFP盒的错误中只有1克隆出3阳性。 条件性敲除（CKO）向量 基因表达的条件消融是调查发育过程或研究生物系统在特定时间点的一个重要工具。有条件的基因敲除的策略通常涉及安置loxP重组位点周围的一个关键的外显子（CE）。当Cre重组酶的表达或活化在CE的缺失产生了移码和成熟前终止密码子，从而导致mRNA的降解，由于无义介导的降解（NMD）。一个条件性基因靶向载体的构建是一个复杂的任务，包括几个步骤亚克隆，靶向和转化22。 SPI提供了一个方便的路线来简化这个过程。作为测试的情况下，Zrsr2基因的等位基因有条件的构建使用SPI方法（ 图5A）。所述Zrsr2基因编码剪接因子和单拷贝位于X染色体上的。基因缺失的条件状态是在这种情况下特别重要的是控制用于 ​​细胞中组成型基因缺失靶向策略适应缺乏Zrsr2期间ES细胞的选择的可能性。 SPI的被执行以与两个不同的LoxP位侧翼的选择盒并发插入亚克隆ZrSr2基因的10kb的一部分。在FRT-PGK-EM7-NEO-FRT-LoxP位纸盒（2 KB）是从PCR扩增RE消化利用滞后链寡核苷酸的保护包含180 bp的HA针对ZrSr2内含子2.含有Rox的-PGK-em7-第二纸盒pL451杀稻瘟（BSD）-Rox-LoxP位（2 KB）的PCR从R6K质粒扩增含180 bp的HA等同于下游地区的内含子3.两侧两个LoxP位点滞后链寡核苷酸的保护外显子3，其D中的CEeletion在时移码有条件的Cre重组酶的激活结果，并引入了一个提前终止密码子。该Zrsr2亚克隆质粒（1.6 KB）的PCR使用含滞后180 bp的HA相匹配的10 kb的Zrsr2序列的末端链寡核苷酸保护的RE线性化P15A ZEO质粒放大。 SPI的反应如前面所述并接种在Zeo的+理学+ BSD板上进行。所述ZrSr2条件靶向载体已成功组装在大多数12重组体检查（ 图5B）的。进一步的DNA测序分析表明，无论是在ZrSr2 CKO载体选择标记的正确插入。 <img alt="图1" src="/files/ftp_upload/52155/52155fig1.jpg" /> 图1。SPI克隆。SPI的克隆过程结合磁带盒插入并亚克隆在单一步骤的概述。 （A）的BAC克隆TRANsformed与pSC101 BADgbaA质粒，在30℃生长。 （B）继阿拉伯糖诱导表达的蛋白质红，不对称修饰插入纸盒和亚克隆质粒引入BAC克隆，并同时与插入和亚克隆标记来生成最终的载体选择。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52155/52155fig1large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图2" src="/files/ftp_upload/52155/52155fig2.jpg" /> 图2示意图示的SPI重组工程寡聚设计的亚克隆质粒和插入盒都通过PCR使用终端PTO和磷酸修饰的寡核苷酸的组合所产生。 PCR片段经红消化在体内产生的ssDNA中间体，其中退火复制叉的后随链。根Ë具体50-180基点HA被纳入各寡，如图所示。箭头指示DNA复制的横跨的候选基因的方向。虚线表示不掺入PCR产物亚克隆质粒序列。稀土部位，最终载体的线性化的限制酶位点;楼前锋序列（20基点），具体到亚克隆质粒或插​​入卡带; R时，质粒或盒的反向互补序列（20碱基对）; SM，选择标记。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52155/52155fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52155/52155fig3.jpg" /> 图3. SPI使施工难度敲入载体。在Dnttip1双重建设使用的SPI策略（一）示意图标记载体。箭INDI凯茨对BAC克隆复制的方向。（B）的电镀的Dnttip1 SPI实验的未诱导和诱导的样品的结果。的Dnttip1 SPI克隆（C）的 EcoRI位摘要。男，1 kb梯度（NEB）; C，P15A Dnttip1缺口修复质粒缺乏的双重标签磁带。片段大小为:标签，9.2 + 4.4 + 2.2 kb的;控制; 9.2 + 4.6 kb的。（D）SPI基于P2rx1-EYFP敲入载体的构建。（E）的EcoRI P2rx1-EYFP SPI克隆摘要。男，1 KB +梯（Invitrogen公司）; C，P15A P2rx1缺口修复质粒缺乏EYFP磁带。片段大小为:标签，8.3 + 4.4 + 3.1 + 0.03 KB;控制; 8.3 + 3.1 + 1.8 + 0.03 KB（F）P2rx1的Southern blot分析- EYFP基因JM8.N4胚胎干细胞的细胞靶向。顶面板，Southern印迹使用PshAI位消化的3&#39;末端的探针。示出的是5个克隆的筛选结果。底板，用南方印迹最终筛选的5&#39;从3确定的积极的结束探头和SpeI消化"。 PshAI位RE网站; S，SpeI位RE网站。虚线表示的矢量的HA的末端。黑盒子代表南部探头。预计限制性片段，PshAI位:WT，8.9 kb的; EYFP-NEO，11.5 kb的; SpeI位:WT，6.9 kb的; EYFP-新 ，7.6 kb的<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52155/52155fig3large.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/52155/52155fig4.jpg" /> 图4。使用SPI。（A）示意图说明BAC的概念，使用SPI 简化修剪修剪BAC。键符号在图1中进行说明。在整个5&#39;和3&#39;（B）的 PCR扩增亚克隆的插入端部，并横跨P2rx1-EYFP insertion个站点。的筛选策略示出了用于每种类型的PCR。 M（上图），Hyperladder 25个基点，（底部面板），1KB +; P，P2rx1 BAC; S，pBeloBAC11 沸石亚克隆质粒（C）的 HindIII位消化从（B）的三个EYFP阳性的SPI BAC克隆; E，预计修剪EYFP BAC的HindⅢ限制性格局。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52155/52155fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52155/52155fig5.jpg" /> 图5.条件性敲除矢量生成采用SPI克隆。（A）中的Zrsr2等位基因的亚克隆中的两个不同的LoxP位侧翼的选择盒同时插入的示意图。键符号在图1中进行说明。（B）的&lt;/对Zrsr2 SPI克隆STRONG&gt;的EcoRI消化。 L，1 kb梯度（NEB）; C1，P15A ZrSr2缺口修复质粒，C2，P15A ZrSr2缺口修复包含新插入片段的质粒; C3，P15A ZrSr2缺口修复包含BSD的插入片段的质粒。片段大小分别是:CKO，7.7 + 2.9 + 2.6 + 1.6 kb的; C1，7.7 + 4.0 kb的; C2，7.7 + 4.3 + 1.6 kb的; C3，7.7 + 2.9 + 2.3 kb的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52155/52155fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>没有步骤</td><td>传统的重组工程管道一 </td><td>多重重组工程B </td></tr><tr><td>第1步</td><td>改造重组工程质粒为BAC主机</td><td>改造重组工程质粒为BAC主机。准备针对磁带和亚克隆载体。 </td></tr><tr><td>步2 </td><td>的R1 / R2 Gateway盒插入</td><td>多重缺口修复克隆</td></tr><tr><td>第3步</td><td>两侧装接loxP侃盒插入</td><td>从单菌落O / N培养</td></tr><tr><td>第4步</td><td>峡修为R3 / R4质粒</td><td>质粒制备和验证</td></tr><tr><td>第5步</td><td>转化酶Cre + E.大肠杆菌 </td><td></td></tr><tr><td>第6步</td><td>质粒制备和验证</td><td></td></tr><tr><td>第7步</td><td> O / N三路网关反应</td><td></td></tr><tr><td>第8步</td><td>三通网关反应进DH10B 大肠杆菌转化大肠杆菌细胞</td><td></td></tr><tr><td>第9步</td><td>从单菌落过夜培养</td><td></td></tr><tr><td>第10步</td><td>质粒制备和序列验证</td><td></td></tr><tr><td合并单元格="3"&gt; 一敲除小鼠计划（KOMP）高通量载体构建管道。 3周的平均时间验证克隆26。 </td></tr><tr><td colspan="2"> 4天B的平均时间为验证克隆</td><td></td></tr></tbody></table> 表1:SPI常规重组工程的条件性敲除载体的构建比较。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> RE缓冲</td><td> 5微升</td></tr><tr><td>脱氧核糖核酸</td><td> 1微克质粒或纯化的PCR产物</td></tr><tr><td> RE </td><td> 1微升（5单位以上） </td></tr><tr><td> TE </td><td>多达50微升</td></tr><tr><td colspan="2">孵育在37℃下至少1小时。根据制造商的说明加热inacitvate </td></tr></tbody></table>表2:RE摘要。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> PCR材料</td><td>终浓度</td></tr><tr><td> PCR缓冲液</td><td> 1X </td></tr><tr><td>的dNTP </td><td> 200纳米</td></tr><tr><td> 硫酸镁 </td><td> 1.5毫米</td></tr><tr><td>甜菜碱</td><td> 130万</td></tr><tr><td> DMSO </td><td> 1％ </td></tr><tr><td>正向引物</td><td> 200纳米</td></tr><tr><td>反向引物</td><td> 200纳米</td></tr><tr><td> DNA聚合酶</td><td> 1 U </td></tr><tr><td>模板</td><td> 10毫微克多拷贝质粒或2.5微升小量的DNA进行基因分型的PCR </td></tr><tr><td>水</td><td>多达50 微升 </td></tr><tr><td colspan="2"> 一个对于具有多拷贝质粒标准的50微升PCR反应。远距离基因分型的PCR分别设立在25μl的PCR反应。</td></tr><tr><td colspan="2"> PCR条件</td></tr><tr><td> 95℃ </td><td> 2分钟</td></tr><tr><td> 92℃ </td><td> 10秒</td></tr><tr><td> 55°C </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 30秒</td></tr><tr><td> 30个循环B </td><td></td></tr><tr><td colspan="2"> B循环中没有可扩展到35，用于BAC PCR基因分型</td></tr></tbody></table> 表3:PCR设置和条件。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>抗生素</td><td> 浓度 （微克毫升-1） </td></tr><tr><td> Ampicllin </td><td> 50 </td></tr><tr><td>灭瘟素B </td><td> 40 </td></tr><tr><td>氯霉素</td><td> 12.5 </td></tr><tr><td>庆大霉素</td><td> 2 </td></tr><tr><td>潮Ç </SUP&gt; </td><td>三十</td></tr><tr><td>卡那霉素B </td><td> 15 </td></tr><tr><td>四环素</td><td> 4 </td></tr><tr><td>甲氧苄胺嘧啶Ç </td><td> 10 </td></tr><tr><td>博来霉素</td><td>五</td></tr><tr><td colspan="2"> 一个在多重重组工程组合使用时，建议与BAC的和多拷贝质粒使用</td></tr><tr><td colspan="2"> B杀稻瘟菌素（35微克毫升-1），卡那霉素（6微克毫升-1）时一起组合使用</td></tr><tr><td colspan="2"> Ç潮霉素和甲氧苄啶不建议选择单拷贝BAC的。 </td></tr></tbody></table> 表4。推荐的抗生素浓度为SPI实验。

Watch this Scientific Journal Video about Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors at JoVE.com

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

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Nanyang Technological University

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JoVE Journal

Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

16.5K 次观看.  University of Leicester. Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

视频: Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

遗传学

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

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 ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

生物工程

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

免疫与感染

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

生物学

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 ...

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

摘要

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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

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

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使用无缝基因组编辑将点突变引入人类多能干细胞

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基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

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