High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.
High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.
复制标题
DOI：
10.1093/nar/gkad1001
复制
发表时间：
2023-12-11
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
携带特定遗传变异的哺乳动物细胞在基础研究和治疗开发方面都显示出巨大的潜力。然而，由于缺乏一种健壮的方法来构建大规模的克隆高质量组合文库，它们的充分利用受到了限制。本研究发展了细胞周期受阻重组酶介导的盒式交换(ARMCE)，能够提供单克隆性、精确的基因组整合和统一的转基因表达。通过优化诺康唑介导的有丝分裂停滞，无需抗生素选择即可获得20%的靶基因替换效率，改进后的aRMCE效率适用于各种受试细胞克隆、转基因靶点和转染法。作为这种通用方法的演示，我们进行了可结晶片段(Fc)的定向进化，为Fc构建了107多个变异体的易出错文库，并在CHO细胞表面显示为Ig G。通过深度测序验证了构建的文库的多样性，并鉴定了一组新的Fc突变体，显示出与特定Fcγ受体结合的改善和增强的效应器功能。由于其运载量大，并与不同的诱变方法兼容，我们期待这一哺乳动物细胞平台技术在定向进化、多重基因分析、细胞系开发和干细胞工程方面有广泛的应用。
Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.
登录
查看更多内容
曲妥珠单抗较低铰链内的单个蛋白水解裂解可降低免疫效应子功能和体内功效。
DOI：
10.1186/bcr3240
发表时间：
2012-08-08
期刊：
Breast cancer research : BCR
影响因子：
--
作者：
Fan X;Brezski RJ;Fa M;Deng H;Oberholtzer A;Gonzalez A;Dubinsky WP;Strohl WR;Jordan RE;Zhang N;An Z
通讯作者：
An Z
影响因子：
14.9
作者：
Anderson, Rachelle P.;Voziyanova, Eugenia;Voziyanov, Yuri
通讯作者：
Voziyanov, Yuri
影响因子：
48
作者：
Joglekar, Alok, V;Leonard, Michael T.;Baltimore, David
通讯作者：
Baltimore, David
影响因子：
21.1
作者：
Chen, Shaopeng;Qiu, Junkang;Hang, Haiying
通讯作者：
Hang, Haiying
影响因子：
64.8
作者：
Anzalone, Andrew V.;Randolph, Peyton B.;Liu, David R.
通讯作者：
Liu, David R.