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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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+cloning+by+homologous+recombination+in+Escherichia+coli.">DNA cloning by homologous recombination in Escherichia coli.</a> Nat. Biotechnol. 18, 1314-1317 (2000).</li><li>Lee, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+Escherichia+coli-based+chromosome+engineering+system+adapted+for+recombinogenic+targeting+and+subcloning+of+BAC+DNA.">A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.</a> Genomics. 73, 56-65 (2001).</li><li>Skarnes, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+knockout+resource+for+the+genome-wide+study+of+mouse+gene+function.">A conditional knockout resource for the genome-wide study of mouse gene function.</a> Nature. 474, 337-342 (2011).</li><li>Valenzuela, D. 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=High-throughput+engineering+of+the+mouse+genome+coupled+with+high-resolution+expression+analysis.">High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.</a> Nat. Biotechnol. 21, 652-659 (2003).</li><li>Bantscheff, 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=Chemoproteomics+profiling+of+HDAC+inhibitors+reveals+selective+targeting+of+HDAC+complexes.">Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes.</a> Nat. Biotechnol. 29, 255-265 (2011).</li><li>Mulryan, K., 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=Reduced+vas+deferens+contraction+and+male+infertility+in+mice+lacking+P2X1+receptors.">Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.</a> Nature. 403, 86-89 (2000).</li><li>Subramanian, K., Rutvisuttinunt, W., Scott, W., Myers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+basis+of+processivity+in+lambda+exonuclease.">The enzymatic basis of processivity in lambda exonuclease.</a> Nucleic Acids Res. 31, 1585-1596 (2003).</li><li>Antoch, 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=Functional+identification+of+the+mouse+circadian+Clock+gene+by+transgenic+BAC+rescue.">Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.</a> Cell. 89, 655-667 (1997).</li><li>Rostovskaya, 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=Transposon-mediated+BAC+transgenesis+in+human+ES+cells.">Transposon-mediated BAC transgenesis in human ES cells.</a> Nucleic Acids Res. 40, e150 (2012).</li><li>Giraldo, P., Montoliu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+use+of+YACs,+BACs+and+PACs+in+transgenic+animals.">Size matters: use of YACs, BACs and PACs in transgenic animals.</a> Transgenic Res. 10, 83-103 (2001).</li><li>Hill, F., 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=BAC+trimming:+minimizing+clone+overlaps.">BAC trimming: minimizing clone overlaps.</a> Genomics. 64, 111-113 (2000).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Cong, L., 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339, 819-823 (2013).</li></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 border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:941px;">Comments/Description</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:315px;">Antibiotics except Zeocin</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">Ampicillin, A9518-5G; 
			Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G; 
			Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Zeocin</td>
			<td style="width:127px;">Invivogen:&#160;</td>
			<td style="width:216px;">ant-zn-1</td>
			<td>Zeocin is also available from Life technologies/Fisher</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">C57BL/6 BAC clones</td>
			<td style="width:127px;">CHORI: https://bacpac.chori.org/&#160;</td>
			<td style="width:216px;">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 height="105">
			<td height="105" style="height:105px;width:315px;">KOD hotstart DNA polymerase system</td>
			<td style="width:127px;">Millipore: https://www.merckmillipore.com</td>
			<td style="width:216px;">71086-3</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">L-Arabinose</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">A3256-25G</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:315px;">Long oligos</td>
			<td style="width:127px;">IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td>
			<td style="width:216px;">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 height="84">
			<td height="84" style="height:84px;width:315px;">MinElute PCR purification kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">28004</td>
			<td>Minimum elution PCR purifications kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">QIAPrep Spin Mini Prep kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">27104</td>
			<td>Silica column or similar matrix kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">Restriction enzymes</td>
			<td style="width:127px;">NEB: https://www.neb.com</td>
			<td style="width:216px;">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/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/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/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/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/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。

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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 ...

subcloning-plus-insertion-spi-novel-recombineering-method-for-rapid

Research

JoVE Journal

Biology

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

16.3K 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

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

科研

生物学

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...