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首页> 外文期刊>Biotechnology and Bioengineering >A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells
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A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells

机译：一种新型BXB1集成酶RMCE系统，用于CHO细胞中MAB表达盒的高保真位点特异性集成

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ABSTRACT <section xml:id="bit26268-sec-0001"> >As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site‐specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO‐S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837–1846. ? 2017 Wiley Periodicals, Inc. </section> </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract XMLNS =“http://www.wiley.com/namespaces/wiley”type =“main”xml：lang =“en”> <title type =“main”>抽象</ title> <section XML：ID =“Bit26268-SEC-0001”> >作为生物治疗生产的CHO细胞系开发通过CHO基因组序列的可用性变得更复杂，准确和可重复地工程师的能力变得越来越重要。用于站点特定集成的多个特征系统将使更复杂的细胞系工程能够生成具有所需属性的细胞系。我们建造并表征了使用BXB1整合酶的新型重组酶介导的盒式交换（RMCE）系统，并将其与常用的FLP / FRT RMCE系统进行比较。我们首先使用CRISPR / CAS9介导的同源重组将由BXB1附着位点或FRT序列（称为着陆垫）侧翼的DNA构建体整合到CHO-S细胞的FER1L4基因组轨迹中。我们的特征在于使用全基因组重构的含有BXB1或FLP / FRT着陆垫的所得克隆，以将其基因组与父母宿主细胞系进行比较。我们确定每个着陆垫在所选克隆中专门集成到FER1L4基因座中，并且由于CRISPR / CAS9修改而观察到基因组或拷贝数的变化中没有主要结构变化。我们随后测试了BXB1和FLP / FRT着陆垫克隆与含有含有BXB1附件位点或FRT位点的相同MAb表达盒的供体载体进行适当的RMCE的能力。我们证明，两个RMCE系统能够在具有可比的MAb表达式类似的时间框架中产生稳定的池。通过遗传表征衍生自任何一个系统的24个克隆，我们确定BXB1 RMCE系统产生比FLP / FRT系统更高的保真度RMCE事件，如克隆更高百分比的克隆，其中MAB盒进入着陆垫进入着陆垫。在各个细胞系中。我们得出结论，BXB1 RMCE是FLP / FRT RMCE的绝佳替代品，对我们的工具箱提供了有价值的补充，从而实现了生物治疗生产更复杂的细胞系的工程。 Biotechnol。生物。 2017; 114：1837-1846。还2017 Wiley期刊，INC。</ p> </段> </ abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15017/'>《Biotechnology and Bioengineering》</a> <b style="margin: 0 2px;">|</b><span>2017年第8期</span><b style="margin: 0 2px;">|</b><span>共10页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Inniss Mara C.&option=202" target="_blank" rel="nofollow">Inniss Mara C.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Bandara Kalpanie&option=202" target="_blank" rel="nofollow">Bandara Kalpanie;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Jusiak Barbara&option=202" target="_blank" rel="nofollow">Jusiak Barbara;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Lu Timothy K.&option=202" target="_blank" rel="nofollow">Lu Timothy K.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Weiss Ron&option=202" target="_blank" rel="nofollow">Weiss Ron;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wroblewska Liliana&option=202" target="_blank" rel="nofollow">Wroblewska Liliana;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Zhang Lin&option=202" target="_blank" rel="nofollow">Zhang Lin;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>Cell Line DevelopmentPfizer IncAndover 01810 Massachusetts;</p> <p>Cell Line DevelopmentPfizer IncAndover 01810 Massachusetts;</p> <p>Synthetic Biology CenterMassachusetts Institute of TechnologyCambridge Massachusetts;</p> <p>Synthetic Biology CenterMassachusetts Institute of TechnologyCambridge Massachusetts;</p> <p>Synthetic Biology CenterMassachusetts Institute of TechnologyCambridge Massachusetts;</p> <p>BioMedicine DesignPfizer IncCambridge Massachusetts;</p> <p>Cell Line DevelopmentPfizer IncAndover 01810 Massachusetts;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/180.html" title="生物工程学（生物技术）">生物工程学（生物技术）;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=CRISPR&option=203" rel="nofollow">CRISPR;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=RMCE&option=203" rel="nofollow">RMCE;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=site‐specific integration&option=203" rel="nofollow">site‐specific integration;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=monoclonal antibody&option=203" rel="nofollow">monoclonal antibody;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=cell line development&option=203" rel="nofollow">cell line development;</a> </p> <div class="translation"> 机译：CRISPR;RMCE;特异性集成;单克隆抗体;细胞系发育; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 class="all_title" id="enpatent66">相似文献</h3> <div class="similaritytab clearfix"> <ul> <li class="active" >外文文献</li> <li >中文文献</li> <li >专利</li> </ul> </div> <div class="similarity_details"> <ul > <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/journal-foreign-detail/0704021881897.html">A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Inniss Mara C.&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Inniss Mara C.,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Bandara 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style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：分枝杆菌噬菌体Bxb1整合酶介导的恶性疟原虫染色体中的有效位点特异性整合</span> </p> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/academic-journal-cn_process-industry_thesis/0201273207160.html">用于mAb收获澄清的高度集成化混合式工艺系统</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wilfried Kappel1&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 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