Trans‐acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high‐throughput microbioreactor system

Hsu Chih‐Jung; Jain Harsh V.; Williams Abasha; Wang Julie; Lute Scott C.; Beaucage Serge L.; Brorson Kurt A.

摘要

Abstract > A rapid and cost‐effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans‐acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High‐throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS 10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS 10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed‐batch serum‐free suspension CHO cell culture; dTtaPS 10+ ‐mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid‐based transfection reagents under similar conditions. The ability of dTtaPS 10+ to deliver plasmids is essentially unaffected by the presence of a silicone‐based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS 10+ ‐plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS 10+ for transient transfection of plasmids into adherent cells and fed‐batch seru </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> > a快速哺乳动物细胞的经济效益瞬态转染方法对于筛查发展的早期发展阶段的生物药物至关重要。为此目的，构建了一种25例两种反式转发寡氧化寡糖硫代胺硫代磷酸酯三磷酸硫磷酰基硫代磷酸酯三酯（DTTTTAPS）转染试剂。使用成像细胞仪和自动微生物反应器系统的文库的高通量筛选导致DTTAPS <SUP> 10 + </ sup>作为有效的转染试剂。该试剂有效地提供了在粘附的HeLa细胞中编码增强的绿色荧光蛋白的质粒，同时表现出低细胞毒性。微生物反应器系统对于评估DTTAPS <SUP> 10 + </ SOP>在FED-批料无血清悬浮液CHO细胞培养中递送PMIMOGloblobulin IgG1的质粒的能力特别有用; DTTAPS 10 + </ sup>介导的转染导致产生的IgG1的产率与在类似条件下的商业脂质的转染试剂中获得的产率相当或更好。 DTTAP 10 + </ sup>递送质粒的能力基本上不受硅氧烷的消泡剂的存在，其通常用于生物反应器细胞培养物中。与在类似条件下使用商业转染试剂复合物的同时实现的同时实现，在水性重构时，冻干DTTTAPS 10 + </ sup> -plasmid复合物的转染效率已经显着恢复。所有实验的结果强调了DTTAPS 10 + </ sup>的电位，用于质粒瞬时转染质粒，进入粘附细胞和奶粉型Seru </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-15016/'>《Biotechnology and Applied Biochemistry》</a> <b style="margin: 0 2px;">|</b><span>2018年第3期</span><b style="margin: 0 2px;">|</b><span>共9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=Hsu Chih‐Jung&option=202" target="_blank" rel="nofollow">Hsu Chih‐Jung;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Jain Harsh V.&option=202" target="_blank" rel="nofollow">Jain Harsh V.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Williams Abasha&option=202" target="_blank" rel="nofollow">Williams Abasha;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wang Julie&option=202" target="_blank" rel="nofollow">Wang Julie;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Lute Scott C.&option=202" target="_blank" rel="nofollow">Lute Scott C.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Beaucage Serge L.&option=202" target="_blank" rel="nofollow">Beaucage Serge L.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Brorson Kurt A.&option=202" target="_blank" rel="nofollow">Brorson Kurt A.;</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>Division of Biotechnology Review and Research II OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research IV OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research II OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research II OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research II OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research IV OBP CDERFood and Drug AdministrationSilver Spring MD USA;</p> <p>Division of Biotechnology Review and Research II OBP CDERFood and Drug AdministrationSilver Spring MD USA;</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><a href="https://www.zhangqiaokeyan.com/clc/177.html" title="生物化学">生物化学;</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=transfection&option=203" rel="nofollow">transfection;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=bioreactors&option=203" rel="nofollow">bioreactors;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=bioprocess&option=203" rel="nofollow">bioprocess;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=CHO cells&option=203" rel="nofollow">CHO cells;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=high‐throughput&option=203" rel="nofollow">high‐throughput;</a> </p> <div class="translation"> 机译：转染;生物反应器;生物过程;CHO细胞;高吞吐量; </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/0704021881211.html">Trans‐acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high‐throughput microbioreactor system</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Hsu Chih‐Jung&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Hsu Chih‐Jung,</a> <a 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<span> <a href="/conference-cn-33950/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 中国化工学会农药专业委员会第九届年会 </a> <span> <span> . 1998</span> </span> </div> </li> <li> <div> <b>7. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020314650469.html">1.L-丝氨酸酯与磷试剂的不对称环化反应用旋光活性磷脂类似物的合成2.S-烷基(芳基)硫代磷(磷)酸衍生物与三氯氧磷的氯化反应的研究</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=陈文彬&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 陈文彬</a> <span> . 1998</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06120112661420.html">一种提高瞬时转染效率的转染试剂及转染方法</a> <b>[P]</b> . <span> 中国专利： CN112695056A </span> <span> . 2021-04-23</span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06120105711997.html">O,O－甲乙基－O－(1－苯基－1,2,4－三唑－3－基)硫代磷酸酯有机磷杀虫,杀螨剂</a> <b>[P]</b> . <span> 中国专利： CN101513187A </span> <span> . 2009-08-26</span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130500160410.html">THROUGHPUT OPTIMIZING REAGENT DISTRIBUTION</a> <b>[P]</b> . <span> 外国专利：  EP2943116B1 </span> <span> . 2021-06-23</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：吞吐量优化试剂分布 </span> </p> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/patent-detail/06130404296529.html">Throughput optimizing reagent distribution</a> <b>[P]</b> . <span> 外国专利：  US10139422B2 </span> <span> . 2018-11-27</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：吞吐量优化试剂分配 </span> </p> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/patent-detail/06130413696259.html">THROUGHPUT OPTIMIZING REAGENT 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